Diterpenoid compounds imparting stress resistance to plants

ABSTRACT

The present invention provites a compound having the following structure: 
     
       
         
         
             
             
         
       
         
         
           
             wherein, in the formula: 
             X is selected from the group consisting of hydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; 
             one of Y 1  and Y 2  is hydrogen or alkyl, and the other is Z-W, wherein Z is a single bond, or a divalent group having alkane or substituted alkane having two hydrogen atoms removed, and W is hydroxy, substituted hydroxy, aldehyde, carboxyl, or substituted carboxyl; and 
             R 1 -R 24  are independently selected from the group of hydrogen, alkyl, and substituted alkyl.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel diterpenoid compounds. Moreparticularly, it relates to novel diterpenoid compounds imparting stressresistance to plants, their uses and methods of synthesizing the same.

2. Background of the Invention

Organisms such as plants are exposed to various stresses (e.g., injury,insect pests, disease and the like) and are required to protectthemselves during their lifespan. Such stresses are highly likely tocause damage which can threaten survival of plants, as compared toorganisms such as animals which can move by themselves.

As used herein, “wound” refers to an injury to a plant caused bycutting, friction, compression, or being fed upon by an insect orHerviora, and physiological functions of the injured sites and broaderregions are disturbed. Wounds due to worms, insects or the like arereferred to as wounds due to an insect pest. In addition to wounds dueto such physical injuries, wounds include those due to air pollutants,or chemical causes such as alkali, acids, heavy metals and the like, aswell as those due to biological causes such as infection by pathogensincluding viruses, bacteria, fungi and the like. Wounds due tomicroorganisms such as viruses, bacteria, fungi or the like areparticularly referred to as disease. Against wounds caused by suchphysical, chemical, or biological causes, organisms such as plants haveparticular defense mechanisms which have adapted themselves to theenvironments that they reside in.

For example, injured plants must immediately cure their wound, recovertheir ability to absorb water etc., from roots, and prevent invasion ofpathogens from the roots. When the tissues are injured, hydrogenperoxide is generated, and lignification, suberization, and oxidativecross-linking of hydroxyproline-rich proteins are started, and cellwalls are repaired and reinforced (Bradley, D. et al. Cell, 79, 21-30,1992). This prevents moisture transpiration from the wound and alsoserves as a physical barrier against the invasion of pathogens. When aplant is wounded, the enzymatic activity of phenylalanine ammonia lyase,which is a rate-limiting enzyme of phenylpropanoid, is elevated and theproduction of polyphenol and lignin, both of which have antibioticactions, are elevated. It is known that the plants infected withpathogens newly produce a series of proteins which are referred to aspathogenesis-related proteins (also referred to as PR proteins). Two ofthe PR proteins is chitinase and −1,3-glucanase and these proteins areproduced when a plant is wounded. It is believed that these enzymesinhibit the growth of bacteria/fungi by degrading chitin and glucan,respectively, both of which are cell wall components of bacteria/fungi,thereby these enzymes protect the plants from infection with pathogensvia the wound.

A protease inhibitor is also produced for the plants' defense againstbeing fed by insects. Protease inhibitor is a general name for amaterial which inhibits an enzyme, spevifically a protease which hasproteolytic activity and is also induced by infection with pathogens.The leaves of tomato and potato plants induce the production of proteaseinhibitor II upon being fed upon by a certain insect pest. If theinsects feed on tissues comprising a large amount of protease inhibitorII, then indigestion is caused and the growth of the insects areinhibited. In addition, the plants protect themselves from insect pestsby attracting a natural enemy etc. For wounds, plants have variousactivities including concentrating substances for the recovery from thewounds by inhibiting photosynthesis other than that for required fordefense, and facilitating cell division for regeneration after the cut.Moreover, it is found that responses against the wound vary depending onfactors such as the extent of the growth, the extent of the wound, theextent of the optical intensity and the like.

In this way, the injured plant generates various responses, however, ithas been revealed that most of the injured plants cause physiologicalresponse by inducing expression of various genes. Of the responses, itis known that jasmonic acid (JA) is involved in the signaling system ina wound-induced response. JA treatment induces the expressions of manywound-induced responsive gene, and thus it is believed that JA is asignal transmitter of the wound-induced response (Creelman, R. A. et al.Plant Cell 9:1211-1223, 1997). JA is synthesized via oxidation oflinolenic acid released from a membrane by lipoxigenase and the like,which is a result of the wounds. In animals, prostaglandin andarachidonic acid metabolites such as leukotrienes which are involved ininflammatory responses are similar to JA and the related substances instructure, and are synthesized from arachidonic acid released from themembrane by phospholipase A₂. In plants as well, it is suggested thatphospholipase A₂ is therefore involved in a liberation of linolenicacid.

Phosphatidic acid, a membrane phospholipid, is liberated from themembrane by wounds and the expression of the wound-induced genes areinduced. It is believed that this liberation results from the hydrolysisof phospholipids, which are membrane components, by phspholipase D(which is moved from the cytoplasm to the membrane byelevated-intracellular Ca²⁺).

When the cell walls are injured, pectin which is a major component ofthe middle lamella in the cell wall is degraded, and oligogalacturonicacid is generated. It is known that the pectin degrading enzyme is alsoinduced by the wounded state.

It is known that MAP kinases, which are some of the serine/threoninetype protein kinase is activated by the wounded state. This activationis found to be induced by phosphorylation. Among MAP kinases activatedby wounds, two kinase a are known: WIPK (wound-induced protein kinase)which is activated at the transcription level and SIPK (salicylicacid-induced protein kinase) which is not controlled by transcription.WIPK of tobacco is also known as a regulator of JA synthesis (Seo, S. etal., Science, 270, 1988-1992, 1995). In the inflammatory response ofanimals, it is known that MAP kinase is involved in the activation ofcytoplasmic phospholipase A₂ (cPLA₂) and thus it is believed that WIPKcontrols JA synthesis by activation of cPLA₂.

Ethylene and abscisic acid (ABA), both of which are phytohormones, arebelieved to be signaling agents in the wound-induced response sinceproduction of these phytohormones is enhanced in the wounded state andtreatments of plants with these phytohormones induces the expression ofwound responsive genes. It is reported that ethylene cooperates with JAand upstream of JA signaling and ABA acts upstream of JA signaling(Dong, X: Curr. Opin. Plant Biol. 1:316-322, 1998; Pelia-Cortes, H. etal. Proc. Natl. Acad. Sci. USA, 92:3106-3114, 1995). Furthermore,ethylene has an effect of accelerating the maturation of fruits and isthus considered a maturation hormone. Also, it is also known thatethylene suppresses extension growth in stems, roots and the like, andaestivation formation, while it accelerates the auxetic growth of stemsand roots, the formation of root hair, the growth of stems in a certainspecies, germination in certain species, epinasty, and the like. JA isalso suggested to be involved in the ubiquitin-proteasome system whichhas various cell control functions.

Wound-induced response is characterized by a systemic response. Forexample, upon injuring leaves, it is found that plants induces such anwound-induced response in not only injured leaves, but also innon-injured leaves. It is thought that in tomatoes the protein systeminparticipates in this systemic wound-induced response. Systemin isexcised from its precursor prosystemin in response to the wounds, todeliver to the entire plant body through seive tubes and induce JAsynthesis.

JA is also suggested to be involved in hypersensitive reaction; HR).Hypersensitive reaction refers to a reaction in which the infected cellsin the plant positively die, thereby confining the pathogen, inhibitingfurther pathogen growth and transition to the entire plant body, andresulting in spot formation. The hypersensitive reaction is believed tobe a kind of disease resistance reaction. As a result of thehypersensitive reaction, the production of not only salicylic acid (SA),but also JA and ethylene is increased. Therefore, hypersensitive celldeath means wound stress. By the hypersensitive response,pathogenesis-related proteins are induced. The pathogenesis-relatedproteins are grouped into acidic pathogen-related proteins and basicpathogenesis-related proteins according to their isoelectric points, theformer are mainly induced by salicylic acid and the latter are mainlyinduced by JA. The acidic pathogenesis-related proteins are poorlyinduced by physical stimulation such as cutting and friction.

It is well known that prostaglandin synthesis is involved ininflammatory responses in animals and is inhibited by salicylic acid oracetyl derivatives thereof, acetyl salicylic acid (also known asaspirin). Also in plants, JA and SA inhibit each others syntheses andfunctions (Niki et al. (1998) Plant Cell Physiol. 39:500-507). The factthat SA and JA, both of which exhibit antagonitic actions, are generatedby the hypersensitive reaction suggests that these two substances finelyregulate signaling in wound-induced responses.

In this way, it is anticipated that JA and SA are known to be related tothe signaling of stresses such as various wounds and that regulation ofthe activity of JA and SA can regulate a resistance to stresses such aswounds.

A number of wound responsive genes have been isolated. The expression ofthese genes is mainly regulated at the transcription level. These genesinclude synthetases, metabolic enzymes, control proteins, defenseproteins and the like.

The mechanisms from injury to expression of the wound responsive genesis considerably different among the genes. For example, for the WIPKgene and the gene encoding 1-aminocyclopropane-t-carboxylic acid (ACC)synthetase which is involved in ethylene synthesis, the accumulation oftranscription products thereof are observed in several minutes to aboutquarter of an hour after the wound. On the other hand, the accumulationof the transcription products of the genes for protein inhibitor II andbasic pathogenesis-related protein become predominant several hoursafter the wound. The accumulation of JA, ethylene and ABA occur within afew or several tens of minutes after the wound. As such, it is suggestedthat other factors may be related to the induction of the expression inWIPK gene an the like. Further, in the induction of the expression ofWIPK gene and the like, other pathways are also predicted, in additionto the pathway via JA.

Therefore, the identification of factors responsible for the regulationof WIPK and SIPK may effectively impart regulation of wound-inducedresponses in organisms such as plants, thus imparting stress resistance.However, heretofore, such factors have not been identified. Therefore,it is desired to seek such factors in the art.

THE PROBLEMS TO BE SOLVED BY THE INVENTIONS

The purpose of the present invention is to impart stress (such as wound)resistance to organisms such as plants, and animals by isolating andsynthesizing the factors involved in WIPK and/or SIPK.

SUMMARY OF THE INVENTION

The above-discussed purpose will be accomplished by isolating orsynthesizing a Labdan-type diterpenoid compound having the followingstructure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

In addition to those described above, the present invention provides thefollowing.

1. A compound having the following structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

2. The compound of item 1, wherein one of Y¹ and Y² is hydrogen, theother is a methylol, substituted methylol, C1-aldehyde, C1-carboxyl, orsubstituted C1-carboxyl group.

3. The compound of item 1, wherein all of R¹-R²⁴ are hydrogen.

4. The compound of claim 1, wherein X is hydroxy.

5. The compound of item 1, having the following structural formula:

6. A composition, comprising a compound having the following structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

7. A composition for imparting stress resistance to a plant oraugmenting said stress resistance, comprising a compound having thefollowing structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

8. The composition of item 7, wherein said stress resistance is at leastone resistance selected from the group consisting of wound resistance,insect resistance, disease resistance, and hypersensitivity cell deathresistance.

9. The composition of item 7, wherein the imparting or augmenting ofsaid stress resistance is accomplished by controlling the activity of atleast one protein selected from the group consisting of wound-inducedprotein kinases, salicylic acid-induced protein kinases,pathogenesis-related proteins, and 1-amino-cyclopropane-t-carboxylicacid synthetases.

10. The composition of item 7, wherein the imparting or augmenting ofsaid stress resistance is accomplished by controlling at least onesignaling system selected from the group consisting of jasmonic acidsignaling systems and salicylic acid signaling systems.

11. A method of imparting stress resistance to a plant or augmentingsaid stress resistance, wherein said method comprises the followingsteps:

a) applying to said plant a compound having the following structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy,. aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

12. The method of item 11, wherein said stress resistance is at leastone resistance selected from the group consisting of wound resistance,insect resistance, disease resistance, and hypersensitivity cell deathresistance.

13. The method of item 11, wherein the imparting or augmenting of saidstress resistance is accomplished by controlling the activity of atleast one protein selected from the group consisting of wound-inducedprotein kinases, salicylic acid-induced protein kinases,pathogenesis-related proteins, and 1-amino-cyclopropane-t-carboxylicacid synthetases.

14. The method of item 11, wherein the imparting or augmenting of saidstress resistance is accomplished by controlling at least one signalingsystem selected from the group consisting of jasmonic acid signalingsystems and salicylic acid signaling systems.

15. A method of producing stress resistant plants, comprising:

a) applying to said plant a compound having the following structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

16. A plant, obtained by the method of item 15.

17. A method of producing stress resistant plant tissues, comprising:

a) applying to said plant tissue a compound having the followingstructure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

18. A plant tissue, obtained by the method of item 17.

19. A method of producing stress resistant plant cells, comprising:

a) applying to said plant cell a compound having the followingstructure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

20. A plant cell, obtained by the method of item 19.

21. A method of producing stress resistant plant seeds, comprising:

a) applying to said plant seed a compound having the followingstructure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

22. A plant seed, obtained by the method of item 21.

23. A method of synthesizing a compound having the following structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl, said method comprises the following steps:

a) reacting a compound (an intermediate 1) having

wherein, in the formula:

R⁵-R²⁴ are independently selected from the group consisting of hydrogen,alkyl, and substituted alkyl, and the same as R¹-R²⁴ for WAF, with alkyllithium to provide an intermediate 2;

b) mixing and reacting the product obtained in a) withm-chloroperbenzoic acid and then with a 10% potassium hydroxide inmethanol to provide an intermediate 4;

c) reacting the product obtained in b) with N-methylmorphorine N-oxidein the presence of tetrapropyl ammonium peruthenate to provide anintermediate 5;

d) adding a compound

wherein, one of V¹ and V² is hydrogen or alkyl, and the other is Z-V,and wherein Z is (CH2)n-C(═O)—O—, V is alkyl, n is an integer of 0 ormore, and R is alkyl, to said intermediate 5 obtained in the step c) inan organic solvent in the presence of NaNH₂ to provide an intermediate(6):

e) adding diisobutyl aluminum hydride in an organic solvent to saidintermediate (6) obtained in the step d) to provide

wherein, X is selected from the group consisting of hydroxy, substitutedhydroxy, halogen, thiol, and substituted thiol;

one of U¹ and U² is hydrogen or alkyl, and the other is Z-U, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and U is hydroxy; and

R¹-R²⁴ are independently selected from the group of 10 hydrogen, alkyl,and substituted alkyl; and optionally

a further oxidation or substitution step where Y¹ is other than hydroxy.

24. The method of item 23, wherein

said X is hydroxy;

one of said Y¹ and Y² is hydrogen, and the other is methylol;

all of R¹-R²⁴ are hydrogen;

said organic solvent is THF;

the alkyl lithium in said step a) is methyl lithium;

one of U¹ and U² is hydrogen, and the other is Z-U, wherein Z is —CH₂—,and U is hydroxy; and

one of said V¹ and V² is hydrogen, and the other is —C(═O)—O—CH₂CH₃.

25. A method of quantifying a compound having the following structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl, said method comprises the following steps:

1) providing a sample;

2) adding the predetermined amount of the steric isomer of said compoundto said sample;

3) separating said sample by a reverse phase column chromatography; and

4) calculating the amount of said compound from said separated stericisomer.

26. The method of item 25, wherein said compound has the followingstructural formula:

said steric isomer has the following structural formula:

27. The method of item 25, wherein said sample is extracted withmethanol and subsequently with methyl acetate, prior to the separationwith said reverse column chromatography.

28. The method of item 25, wherein the separation with said reversecolumn chromatography comprises a separation with a C18 reverse columnchromatography, and said separation comprises a first separation in80%:20% (v/v) methanol:water, and a separation with 9:8 (v/v)acetonitrile:water.

29. The method of item 25, wherein said calculation comprises thecorrection of the recovery loss.

30. A composition for inducing a rapid accumulation of a WRKY familygene in a plant under a condition requiring the accumulation of a WRKYfamily gene, said composition comprises a compound having the followingstructure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

31. The composition of item 30, wherein said compound has the followingstructural formula:

32. The composition of item 30, wherein the condition requiring theaccumulation of said WRKY family gene is a condition requiring the rapidresponse to stress.

33. The composition of item 30, wherein said plant is provided with awound resistance, insect resistance, disease resistance, andhypersensitivity cell death resistance by inducing a rapid accumulationof said WRKY family gene.

34. The composition of item 30, wherein said WRKY family gene is WIZZ orTIZZ.

35. A composition for regulating the expression of a WRKY family gene,comprising a compound having the following structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

36. A method of inducing a rapid accumulation of a WRKY family gene in aplant under a condition requiring the accumulation of a WRKY familygene, wherein said method comprises the following steps:

a) applying to said plant a compound having the following structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

37. The method of item 36, wherein said compound has the followingstructural formula:

38. The method of item 36, wherein the condition requiring theaccumulation of said WRKY family gene is a condition requiring the rapidresponse to stress.

39. The method of item 36, wherein said plant is provided with a woundresistance, insect resistance, disease resistance, and hypersensitivitycell death resistance by inducing a rapid accumulation of said WRKYfamily gene.

40. The method of item 36, wherein said WRKY family gene is WIZZ orTIZZ.

41. The method of item 36, wherein said compound is applied immediatelyafter the accumulation of said WRKY family gene is required.

42. A composition for regulating the expression of a WRKY family gene,comprising the compound of item 1.

43. A use of a compound for imparting stress resistance to a plant oraugmenting said stress resistance, said compound having the followingstructure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

44. A use of a compound for producing stress resistant plants, saidcompound having the following structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

45. A use of a compound for producing stress resistant plant tissues,said compound having the following structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

46. A use of a compound for producing stress resistant plant cells, saidcompound having the following structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

47. A use of a compound for producing stress resistant plant seeds, saidcompound having the following structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

48. A use of a compound for inducing a rapid accumulation of a WRKYfamily gene in a plant requiring the accumulation of said WRKY familygene, said compound having the following structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

49. A use of a compound for regulating the expression of a WRKY familygene, said compound having the following structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

50. A composition for facilitating the elongating growth or auxeticgrowth of a plant, inhibiting the elongating growth of a plant,facilitating the maturation of a plant, or regulating the flowering of aplant, said composition comprises a compound having the followingstructure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

51. A method of facilitating the elongating growth or auxetic growth ofa plant, inhibiting the elongating growth of a plant, facilitating thematuration of a plant, or regulating the flowering of a plant, saidmethod comprises applying to a plant a compound having the followingstructure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

52. A plant, obtained by the method of item 51.

53. A use of a compound for facilitating the elongating growth orauxetic growth of a plant, inhibiting the elongating growth of a plant,facilitating the maturation of a plant, or controlling the flowering ofa plant, said method comprises applying to a plant a compound having thefollowing structure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, and substituted thiol;

one of Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chromatogram upon isolating the compound of theinvention.

In FIG. 2, FIG. 2A shows the effects of the compound of the invention onthe induction of WIPK activity. The isolated compound at theconcentrations indicated was absorbed into the subject through thepetiole and WIPK activity was measured after 15 minutes. As a controlgroup, a buffer only was absorbed into the subject. As another controlgroup (healthy leaves), leaves were cut from the tobacco plant body andwere subjected to WIPK activity measurement immediately. FIG. 2B showsthe result of WIPK activity (MBP phosphorylation) that was measured 5min, 15 min, and 30 min after the naturally isolated substances at someconcentrations (1 nM, 5 nM and 10 nM) and water as control are givenfrom petiole to leaves. The induction of WIPK activity was observed at 5min and maximum activation at 15 min and it was found that maximumactivity is maintained even after 30 min.

FIG. 3 shows the effect of the compound of the invention on theinduction of the expression at the PI-II gene. The isolated compound atthe concentrations indicated was absorbed into the subject through thepetiole and a transcription product of PI-II gene is measured after acertain period of time. As control group, a buffer only was given.

FIG. 4 shows the effect of the compound of the invention on theinduction of SIPK activity. The isolated compound at the concentrationsindicated was absorbed into the subject through the petiole and SIPKactivity was measured after 15 minutes. As a control group, a bufferonly was given. Also, as another control (healthy leaves), leaves werecut from the tobacco plant body and were subjected to SIPK activitymeasurement immediately.

FIG. 5 shows the effect of the synthesized compound of the invention onthe induction of WIPK activity. MBP indicates the intensity of MBPphosphorylation.

FIG. 6 shows the effect of the synthesized compound of the invention onthe induction of SIPK activity. MBP indicates the intensity of MBPphosphorylation.

FIG. 7 shows an examination in WIPK and SIPK activities of the naturalactive substance of the invention.

In FIG. 8, FIG. 8A shows an endogenous accumulation of the activesubstance of the invention after transfer from 20° C. to 30° C. FIG. 8Bshows an endogenous accumulation of the active substance of theinvention after initiating incubation at 20° C.

FIG. 9 shows an endogenous accumulation of the active substance of theinvention after wounding.

FIG. 10 shows the induction of WIZZ transcription level by the compoundof the invention. WIZZ indicates the induction of WIZZ by the compoundof the invention and Actin indicates the induction when using actin ascontrol (Control (0 min), 30 min after treatment by water or WAF-1).

FIG. 11 shows the ethylene level released from tobacco leaves whichreceived the compound of the invention.

In FIG. 12, FIG. 12A shows the effect on the defense of tobacco leavesagainst TMV infection shown as size of necrosis spots, when the compoundof the invention at 10 pM to 100 nM concentrations or water was given tothe leaves. FIG. 12B shows pictures which exhibit the defense of tobaccoleaves against TMV infection when the compound of the invention at 100nM concentrations or water was given to the leaves. FIG. 12C shows thecomparison of amounts of TMV coat protein when the compound of theinvention at 100 pM or 100 nM, or water was given to the tobacco leaves.

PREFERRED EMBODIMENTS OF THE INVENTION

The following illustrates the present invention. It should be understoodthat the singular forms include plural forms throughout thespecification unless otherwise indicated. Thus, it should be understoodthat a singular article (for example “a,” “an”, “the” and the like forEnglish, “ein”, “der”, “das”, “die” and the like, and their declinedforms for German, “un”, “une”, “le”, “la” and the like for French, “un”,“una”, “el”, “la” and the like for Spanish, and corresponding articlesand adjectives for other languages) include their plural forms unlessotherwise indicated. It should be understood that the terms as usedherein refer to those meanings commonly used in the art unless otherwiseindicated.

(Terms)

Definitions of the terms as specifically used herein are listed below.

As used herein, the term “organism” is used in the broadest meaning inthe art and refers to objects which maintain a living state, whichtypically have various characteristics such as cellular structure,growth (self-proliferation), growth, regulation, metabolism, repairingability, and the like, as basic properties, normally have inheritancethat nucleic acids control and proliferation involved in metabolism thatproteins control. Organisms include prokaryotes, eukaryotes (such asplants, animals) and the like.

As used herein, the term “plant(s)” is a general name of organismsbelonging to Plantae and characterized by immotile organisms which havechlorophyl, hard cell walls, and presence of abundant continuousembryonic tissue. Typically, plants refers to flowering plants havingcell walls, and anabolism by chlorophyll. “Plants” include any of themonocotyledons and the dicotyledons. Preferred plants includemonocotyledons belonging to Gramineae such as wheat, maize, rice,barley, and sorghum. Other examples of preferred plants include tobacco,piment, eggplant, melon, tomato, sweat potato, cabbage, cibol, broccoli,carrot, cucumber, citrus, celery cabbage, lettuce, peach, potato, andapple. In addition to crop plants, preferred plants include, but are notlimited to, flowering plants, trees, grasses, weeds and the like. Unlessindicated otherwise, plants mean any of the plants bodies, the plant'sorgans, the plant's tissues, plant's cells, and the plant's seeds.Example of plant's organs includes roots, leaves, stems, and flowers.Example of the plant's cells includes callus and cells in suspensionculture.

In other embodiments, examples of plant species which maybe used in theinvention includes plants of Solanaceae, Gramineae, Cruciferae,Rosaceae, Leguminosae, Cucurbitaceae, Labiatae, Liliaceae,Chenopodiaceae, Umbelliferae.

Examples of Solanaceae include plants belonging to Nicotiana, Solanum,Datura, Lycopersion, or Petunia (including e.g., tobacco, aubergines(eggplant), potatoes, tomatoes, capsicums, petunias and the like).

Examples of Gramineae include plants belonging to Oryza, Hordenum,Secale, Scccharum, Echinochloa, or Zea (including e.g., rice, barley,rye, cockspur, sorghum, maize and the like.

Examples of Cruciferae include plants belonging to Raphanus, Brassica,Arabidopsis, Wasabia, or Capsella (including e.g., Chinese radish,mustard, thalecress, Japanese horse radish, shepherds's purse and thelike.

Example of Rosaceae includes plants belonging to Orunus, Malus, Pynus,Fragaria, or Rosa (including e.g., Japanese apricots, peaches, apples,pears, strawberries, roses and the like).

Examples of Leguminosae include plants belonging to Glycine, Vigna,Phaseolus, Pisum, Vicia, Arachis, Trifolium, Alphalfa, or Medicago(including e.g., soy beans, azuki beans, kidney beans, peas, broadbeans, peanuts, clovers, bur clovers and the like).

Examples of Cucurbitaceae include plants belonging to Luffa, Cucurbita,or Cucumis (including e.g., dishcloth gourds, pumpkins, cucumbers,melons and the like).

Examples of Labiatae include plants belonging to Lavandula, Mentha, orPerilla (including e.g., lavenders, mints, beefsteak plants and thelike).

Examples of Liliaceae include plants belonging to Allium, Lilium, orTulipa (including e.g., cibols, garlics, lilies, tulips and the like).

Example of Chenopodiaceae includes plants belonging to Spinacia(including e.g., spinach).

Examples of Umbelliferae includes plants belonging to Angelica, Daucus,Cryptotaenia, or Apitum (including e.g., archangels, carrots, Japanesehornworts, celeries and the like).

The plants used in the present method are preferably tobaccos, tomatoes,potatoes, rices, maizes, Chinese radishs, soy beans, peas, bur clovers,and spinaches, more preferably, tobaccos, tomatoes, potatoes, maizes,and soybeans.

The present agent may be useful for imparting stress resistance tovarious organisms including animals as well as plants. As used herein,the term “Animal(s)” are used in the broadest meaning in the art,including vertebrate and invertebrate. Animals includes, but is notlimited to, Mammalia, Aves, Reptilia, Amphibia, Pisciformes, Insecta,Vermes and the like. Therefore, organisms and animals in thespecification include every organism that may be stressed.

As used herein, the term “stress” refers to factors which mayphysically, chemically, or biologically impart stress to an organismsuch as a plant, and/or prevent normal growth/proliferation of theorganism. Stress includes physical stress (such as the result of light,heat, cooling, freezing, ultraviolet radiation, X-rays, cutting,friction), chemical stress (such as the result of the oxygen stress,chemical substances, bioactive substance)) biological stress (such asthe result of viruses, pathogens (including e.g., infection with apathogen which causes rice blast in plants), for example.Characteristics for the expression of genes including genes involved instress may be determined by extracting RNA from any part of an organismsuch as a plant to analyze amounts of the expression by Northern blotanalysis or quantitate expressed proteins by Western blot analysis.

Therefore, as used herein, the term “stress resistance” refers todelaying, stopping, or recovering a reaction which normally weakendorganisms such as plants, or otherwise not generating the reaction whichwould weaken otherwise the organisms. Thus, the organisms to whichstress resistance is imparted have an elevated survival ratio comparedto organisms to which stress resistance not imparted. As used herein,“impart stress resistance” refers to maintaining the organisms (whereinthe organisms are treated) in the living state, whereas wild-typeorganisms would otherwise completely or almost completely die when somestress is imparted. Such survival can be examined using techniques wellknown in the art. For example, survival can be examined with the nakedeye or via microscope. As used herein, “augment stress resistance”refers to increasing a resistance to a stress in treated organismswhereas the wild-type organisms show some extent of resistance uponreceiving the stress. Since imparting and augmenting stress resistancesometimes may have meanings that overlap one another, these terms may beused interchangeably herein.

As used herein, the term “wound resistance” refers to a property oforganisms such as plants and the ability to reduce the extent of awound. As used herein, the term “disease resistance” refers to aproperty of organisms such as plants and the ability to reduce an extentof disease development. As used herein, the term “insect resistance”refers to a property of organisms such as plants and the ability toreduce harm upon being fed upon. Therefore, “stress resistance” as usedherein encompasses “wound resistance”, “insect resistance”, and “diseaseresistance”.

It is contemplated that agents, compounds, compositions and methods ofthe invention function in not only monocotyledons, but also dicotyledonsand other organisms including animals. This is explained by the basicmechanisms of stress responsive regulation being similar betweenmonocotyledons and dicotyledons and the inflammatory responses inanimals have similar mechanisms (arachidonic acid metabolic pathway) aswell.

As used herein, the term “elongating growth” refers to apical growth,intercalary growth and the like. “Apical growth” is conducted at agrowing point in apical stems or apical roots and refers to theformation of axial organs such as stems and roots. “Intercalary growth”refers to that when enlongating, the growth does not occur equally ineach part, but occurs in growth region between grown parts (e.g., organsor tissues, in which growth has been stopped) to some extent and alsorefers to internodal growth.

As used herein, the term “auxetic growth” refers to growth within thevolume of the plant body, plant tissue or plant cell which increasesmass. In the plant tissues or organs, auxetic growth also generates anincreased number of plant cells.

As used herein, “maturation” refers to a process from the time of germcell maturation in the plant body or plant tissue or the completion ofthe growth period of the fruit body to complete maturation.

As used herein, “regulating flowering” refers to changing a time periodfrom an aestivation formation to an actual flowering.

Herein, cultivation of plants can be carried out by any of the methodsknown in the art. Cultivating methods of plants are exemplified in forexample, “Experimental protocols in model plants-rice/Arabidopsis-”:Saibo-kogaku Bessatu series (Cellular Engineering Supplemental);“Ine-no-saibai-ho (Cultivation method for rice)” (Kazutoshi Okuno) pp.28-32, and “Arabidopsis-no-baiyou-ho (Cultivation method forArabidopsis)” (Yasuo Niwa) pp. 33-40(Eds. Ko Shimamoto, Kiyotaka Okada)and thus those skilled in the art can easily carry out the methods andit is not necessary to describe such method herein in detail. Forexample, cultivation of Arabidopsis can be performed by any of themethod of geoponics, rock wool cultivation, and hydroponics. If it iscultivated under continuous light condition using a white fluorescentlamp (about 6000 lux), Arabidopsis begins to bloom 4 weeks after seedingand produces mature seeds 16 days after flowering. 40 to 50 seeds perpod are obtained and 10000 seeds are obtained in 2 to 3 months beforedeath. The dormancy period of the seed is short, after drying the seedsfor about 1 week, matured seeds germinate in 2 to 3 days after absorbingwater. If the seeds are treated at a low temperature of 4° C. afterabsorbing water and seeding, germination is synchronized. Cultivation ofrice is conducted in geoponics, grown under a light condition of 10000lux or more. Transferring the conditions to short day condition, resultsin heading induced about 40 days after seeding, the plants bloom 30 daysafter heading and matured seeds are obtained about 40 days afterflowering.

For culturing, differentiating and regenerating plant cells, theprocedures and media known in the art are used. Such media include, butare not limited to Murashige-Skoog (MS) medium, GaMborg B5 (B) medium,White medium, Nitsch & Nitsch (Nitsch) medium and the like. These mediaare normally used with the addition of a suitable amount of plant growthregulation substances (phytohormones) and the like. Preferably, theagent of the invention is used as an additive in the media and may serveas a regulator or growth (proliferation), differentiation andregeneration of plant body, plant tissue or organ, or plant cells. Also,the agent of the invention is not only comprised as an additive for themedia, but also comprised in various forms (e.g., agriculturalcompositions such as a solid fertilizer, a liquid nutrition agent andthe like) as described below.

Analysis of the expression regulation in WIPK- or SIPK-associated genesand the like can be conducted by gene analysis method using DNA arrays.DNA arrays are generally reviewed in Saibo-kogaku Bessatu (CellularEngineering Supplemental) “DNA microarray-to-saisin PCR-ho (DNAmicroarray and Current PCR method)”, Ed., Shujunsha Co. Ltd. Analysis ofplants using DNA array has recently been conducted (Schenk P M et al.,(2000) Proc. Natl. Acad. Sci. (USA) 97:11655-11660). Hereinafter, DNAarrays and genetic analysis methods using the same are brieflyexplained.

“DNA array” refers to a device in which DNAs are arrayed and immobilizedon a plate. DNA arrays are divided into DNA macroarrays, DNAmicroarrays, and the like according to the size of the plate or thedensity of DNA placed on the plate.

The border between macro and micro is not strictly determined. However,generally, “DNA macroarray” refers to a high density filter in which DNAis spotted on a membrane, while “DNA microarray” refers to a plate ofglass, silicon, and the like which carries DNA on a surface thereof.There are a cDNA arrays, oligoDNA arrays, and the like varying accordingto the type of DNA placed on the array.

A certain high density oligoDNA array, in which a photolithographytechnique for production of semiconductor integrated circuits isutilized and a plurality of oligoDNAs are simultaneously synthesized ona plate, is specifically called a “DNA chip”, an adaptation of the term“semiconductor chip”. Examples of the DNA chip prepared by this methodinclude GeneChip® (Affymetrix, CA), and the like (see Marshall A et al.,(1998) Nat. Biotechnol. 16:27-31 and Ramsay G et al., (1998) Nat.Biotechnol. 16 40-44. Preferably, GeneChip® may be used in geneticanalysis using a microarray according to the present invention. The DNAchip is defined as described above in a narrow sense, but may refer toall types of DNA arrays or DNA microarrays.

Thus, DNA microarrays are devices in which several thousands to severalten thousands or more of gene DNAs are arrayed on a glass plate in highdensity. Therefore, it is made possible to analyze gene expressionprofiles or gene polymorphism on a genomic scale by hybridization ofcDNA, cRNA or genomic DNA. With this technique, it has been madepossible to analyze a signaling system and/or a transcription controlpathway (Fambrough D et al., (1999), Cell 97,727-741); the mechanism oftissue repair (Iyer V R et al., (1999), Science 283:83-87); the actionand mechanism of medicaments (Marton M J, (1999), Nat. Med.4:1293-1301); fluctuations in gene expression during development anddifferentiation processes on a wide scale; identify a gene group whoseexpression fluctuates according to pathologic conditions and the like;find a novel gene involved in a signaling system or a transcriptioncontrol; and the like. Further, as to gene polymorphism, it has beenmade possible to analyze a number of SNPs with a single DNA microarray(Cargill M et al., (1999), Nat. Genet. 22:231-238).

As a labeling method for synthesized DNA arrays, for example, doublefluorescence labeling is used. In this method, two different mRNAsamples are labeled by different respective fluorescent dyes. The twosamples are subjected to competitive hybridization on the samemicroarray, and both fluorescences are measured. By comparing thefluorescences, differences in gene expression can be detected. Examplesof the fluorescent dye include, but are not limited to, Cy5 and Cy3,which are most often used, and the like. The advantage of Cy3 and Cy5 isthat the wavelengths of fluorescences do not overlap substantially.Double fluorescence labeling may be used to detect mutations ormorphorisms in addition to differences in gene expression.

In assays using a DNA array, a fluorescent signal indicatinghybridization on the DNA microarray is detected by a fluorescencedetector or the like. As such a detector, there are conventionallyvarious available detectors. For example, a research group at theStanford University has developed an original scanner which is acombination of a fluorescence microscope and a movable stage (seehttp://cmgm.stanford.edu/pbrown). A conventional fluorescence imageanalyzer for gels, such as FMBIO (Hitachi Software Engineering), Storm(Molecular Dynamics), and the like, can read a DNA microarray if thespots are not arrayed in too great a density. Examples of otheravailable detectors include ScanArray 4000 and 5000 (GeneralScanning;scan type (confocal type)), GMS418 Array Scanner (Takara Shuzo; scantype (confocal type)) Gene Tip Scanner (Nippon Laser&Electronics Lab.;scan type (non-confocal type)), Gene Tac 2000 (Genomic Solutions; CCDcamera type)), and the like.

The amount of data obtained from DNA microarrays is huge. Software formanaging correspondences between clones and spots, analyzing data, andthe like is important. As such software, software attached to eachdetection system is available (Ermolaeva O et al., (1998) Nat. Genet.20:19-23). Further, an example of a database format is GATC (geneticanalysis technology consortium) proposed by Affymetrix.

The regulation of the expression in WIPK- and SIPK-related genes as wellas the down stream genes thereof by the agent, compound or compositionof the invention may also be used in genetic analysis using adifferential display technique.

Differential display techniques are methods for detecting or identifyinggenes whose expression fluctuates. In this method, cDNA is prepared fromeach of at least two samples, and amplified by PCR using a set of anyprimers. Thereafter, a plurality of generated PCR products are separatedby gel electrophoresis. After the electrophoresis pattern is produced,genes with fluctuating expression are cloned based on relative signalstrength change between each band.

In this way, techniques that regulate expression of the genes upstreamor downstream of the signaling pathway regulated by the compound orsubstance of the invention are well known.

The structure of the compound according to the invention can bedetermined by techniques well known in the art. Such methods fordetermining the structure includes, but is not limited to, physicalanalysis methods such as NMR, X-ray structural analysis, IR analysis,mass spectrometry; chemical analysis methods such as method usingspecific chemical reactions with particular substituents, like theFehling reaction; biochemical analysis using a particular enzyme, suchas aldehyde dehydrogenase, which specifically reacts with a particularsubstitute; biological analysis using microorganisms and the like.Preferably, physical analysis methods such as NMR are used.

(Organic Chemistry)

It is to be understood that any compound as used herein includes anyisomers thereof, for example, structural isomers, steric isomers whichhave the same structural formula, but different atomic conformations andconfigurations (e.g., geometric isomers, atrop isomers, opticalisomers), and cis-trans isomers (such as (E,E) (E,Z), (Z,E), (Z,Z), andthe like, racemate, enantiomers and the like). In one embodiment, thecompound of the present invention as used herein maybe a single isomer,or a mixture of two or more isomers.

As used herein, the term, “diterpenoid” refers to a compound having atwo consecutive terpenoid structures. A terpene is a generic designationthat refers to an organic compound from various plants (or in rarecases, from animals) having a carbon number of a multiple of five (5n;n≧2) and biosynthetically derived from precursors having n isoprene orisopentane units. A terpene is also designated as a terpenoid. Variousterpene-type hydrocarbons such as alcohols, ketones, aldehydes,carboxylic acids, lactones, and the like are known in the art. Terpenesare classified as follows according to their carbon numbers. Amongthese, those having n=4 are designated as diterpenes (having a carbonnumber of 20).

As used herein, the term, “Labdan-type” refers to a compound having thefollowing (labdan) structure:

The Labdan-type compound of the present invention may have anysubstituent. The substituents include any substituents selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, alkynyl, substituted alkynyl, alkoxy,substituted alkoxy, carbocyclic groups, substituted carbocyclic groups,heterocyclic groups, substituted heterocyclic groups, halogen, hydroxy,substituted hydroxy, thiol, substituted thiol, cyano, nitro, amino,substituted amino, carboxy, substituted carboxy, acyl, substituted acyl,thiocarboxy, substituted thiocarboxy, amide, substituted amide,substituted carbonyl, substituted thiocarbonyl, substituted sulfonyl,and substituted sulfinyl, but those substituents which do not affect thehydrophilicity, polarity, and the like are preferable. Such substituentsinclude those substituents selected from the group consisting ofhydrogen, alkyl, and substituted alkyl. Preferably, the alkyl may beC1-C6 alkyl, more preferably C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl,C1-C2 alkyl, methyl, and the like. When the substituted alkyl isselected as the substituent, preferably, the substituted portion may asubstituent which does not affect the hydrophilicity, polarity, and thelike.

Also, the substituent may be a substituent selected from the groupconsisting of halogen, alkoxy, substituted alkoxy, amino, substitutedamino, alkylthio, substituted alkylthio, arylthio, substituted arylthio,nitro, carboxy, substituted carboxy, acyl, substituted acyl, andsubstituted sulfonyl, so long as it does not affect the essentialfunction of the present invention.

As used herein, the term, “alkyl” refers to a monovalent group havingone hydrogen atom removed from an aliphatic hydrocarbon (alkane) such asmethane, ethane, propane, and the like, and is generally represented byC_(n)H_(2n+1)— (wherein n is a positive integer). The alkyl may bestraight chain or branched. The term, “substituted alkyl” refers to analkyl having its H substituted with the following substituent. Specificexamples of these may include C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl,C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10alkyl, C1-C11 alkyl, or C1-C12 alkyl, C1-C2 substituted alkyl, C1-C3substituted alkyl, C1-C4 substituted alkyl, C1-C5 substituted alkyl,C1-C6 substituted alkyl, C1-C7 substituted alkyl, C1-C8 substitutedalkyl, C1-C9 substituted alkyl, C1-C10 substituted alkyl, C1-C11substituted alkyl, or C1-C12 substituted alkyl. For example, C1-C10alkyl refers to a straight chain or branched alkyl having 1-10 carbonatoms, and is represented by methyl (CH₃—), ethyl (C₂H₅—), n-propyl(CH₃CH₂CH₂—), isopropyl ((CH₃)₂CH—) n-butyl (CH₃CH₂CH₂CH₂—), n-pentyl(CH₃CH₂CH₂CH₂CH₂—), n-hexyl (CH₃CH₂CH₂CH₂CH₂CH₂—), n-heptyl(CH₃CH₂CH₂CH₂CH₂CH₂CH₂—), n-octyl (CH₃CH₂CH₂CH₂CH₂CH₂CH₂CH₂—), n-nonyl(CH₃CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—), n-decyl(CH₃CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—), —C(CH₃)₂CH₂CH₂CH(CH₃)₂, —CH₂CH(CH₃)₂,and the like. Also, for example, the C1-C10 substituted alkyl refers toa C1-C10 alkyl having its one or more hydrogen atoms substituted with asubstituent.

As used herein, the term, “alkane” refers to an aliphatic hydrocarbonrepresented by the general formula C_(n)H_(2n+2). The alkanes includemethane, ethane, propane, and the like. The alkane may be straight chainor branched. The term, “substituted alkane” refers to an alkane havingits H substituted with the following substituent. Specific examples ofthese may include C1-C2 alkane, C1-C3 alkane, C1-C4 alkane, C1-C5alkane, C1-C6 alkane, C1-C7 alkane, C1-C8 alkane, C1-C9 alkane, C1-C10alkane, C1-C11 alkane, or C1-C12 alkane, C1-C2 substituted alkane, C1-C3substituted alkane, C1-C4 substituted alkane, C1-C5 substituted alkane,C1-C6 substituted alkane, C1-C7 substituted alkane, C1-C8 substitutedalkane, C1-C9 substituted alkane, C1-C10 substituted alkane, C1-C11substituted alkane, or C1-C12 substituted alkane. For example, C1-C10alkane refers to a straight chain or branched alkane having 1-10 carbonatoms, and is represented by methane (CH₄), ethane (C₂H₆), n-propane(CH₃CH₂CH₃), isopropane ((CH₃)₂CH₂), n-butane (CH₃CH₂CH₂CH₃), n-pentane(CH₃CH₂CH₂CH₂CH₃), n-hexane (CH₃CH₂CH₂CH₂CH₂CH₃), n-heptane(CH₃CH₂CH₂CH₂CH₂CH₂CH₃), n-octane (CH₃CH₂CH₂CH₂CH₂CH₂CH₂CH₃), n-nonane(CH₃CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₃), n-decane(CH₃CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₃), CH(CH₃)₂CH₂CH₂CH₂CH(CH₃)₂,CH₃CH(CH₃)₂, and the like. Also, for example, the C1-C10 substitutedalkane refers to a C1-C10 alkane having its one or more hydrogen atomssubstituted with a substituent. Therefore, as used herein, the term, “adivalent group having alkane or substituted alkane having two hydrogensremoved” refers to a divalent group created by removing any twohydrogens from the above-described alkane or substituted alkane. Thesubstituent is also designated as an alkylene group. These substituentsinclude, but are not limited to, trimethylene (—(CH₂)₃—), tetramethylene(—(CH₂)₄—), pentamethylene (—(CH₂)₅—), and the like.

As used herein, the term, “cycloalkyl” refers to an alkyl having acyclic structure. The term, “substituted cycloalkyl” refers to acycloalkyl having its H substituted with the following substituent.Specific examples of these may include C3-C4 cycloalkyl, C3-C5cycloalkyl, C3-C6 cycloalkyl, C3-C7 cycloalkyl, C3-C8 cycloalkyl, C3-C9cycloalkyl, C3-C10 cycloalkyl, C3-C11 cycloalkyl, C3-C12 cycloalkyl,C3-C4 substituted cycloalkyl, C3-C5 substituted cycloalkyl, C3-C6substituted cycloalkyl, C3-C7 substituted cycloalkyl, C3-C8 substitutedcycloalkyl, C3-C9 substituted cycloalkyl, C3-C10 substituted cycloalkyl,C3-C11 substituted cycloalkyl or C3-C12 substituted cycloalkyl. Forexample, the cycloalkyl is represented by cyclopropyl, cyclohexyl, andthe like.

The term, “alkenyl” refers to a monovalent group having one hydrogenatom removed from an aliphatic hydrocarbon having one double bond withinthe molecule, such as ethylene, propylene, and the like, and isgenerally represented by C_(n)H_(2n−1)— (wherein n is a positive integerof two or more). The term, “substituted alkenyl” refers to an alkenylhaving its H substituted with the following substituent. Specificexamples may include C2-C3 alkenyl, C2-C4 alkenyl, C2-C5 alkenyl, C2-C6alkenyl, C2-C7 alkenyl, C2-C8 alkenyl, C2-C9 alkenyl, C2-C10 alkenyl,C2-C11 alkenyl, or C2-C12 alkenyl, C2-C3 substituted alkenyl, C2-C4substituted alkenyl, C2-C5 substituted alkenyl, C2-C6 substitutedalkenyl, C2-C7 substituted alkenyl, C2-C8 substituted alkenyl, C2-C9substituted alkenyl, C2-C10 substituted alkenyl, C2-C11 substitutedalkenyl, or C2-C12 substituted alkenyl. For example, C2-C10 alkenylrefers to a straight chain or branched alkenyl having 2-10 carbon atoms,and is represented by vinyl (CH₂═CH—), allyl (CH₂═CHCH₂—), CH₃CH═CH—,and the like. Also, for example, the C2-C10 substituted alkenyl refersto a C2-C10 alkenyl having its one or more hydrogen atoms substitutedwith a substituent.

As used herein, the term, “cycloalkenyl” refers to an alkenyl having acyclic structure. The term, “substituted cycloalkenyl” refers to acycloalkenyl having its H substituted with the following substituent.Specific examples of these may include C3-C4 cycloalkenyl, C3-C5cycloalkenyl, C3-C6 cycloalkenyl, C3-C7 cycloalkenyl, C3-C8cycloalkenyl, C3-C9 cycloalkenyl, C3-C10 cycloalkenyl, C3-C11cycloalkenyl, C3-C12 cycloalkenyl, C3-C4 substituted cycloalkenyl, C3-C5substituted cycloalkenyl, C3-C6 substituted cycloalkenyl, C3-C7substituted cycloalkenyl, C3-C8 substituted cycloalkenyl, C3-C9substituted cycloalkenyl, C3-C10 substituted cycloalkenyl, C3-C11substituted cycloalkenyl, or C3-C12 substituted cycloalkenyl. Forexample, the preferred cycloalkenyl is represented by 1-cyclopentenyl,2-cyclohexenyl, and the like.

The term, “alkynyl” refers to a monovalent group having one hydrogenatom removed from an aliphatic hydrocarbon having one triple bond withinthe molecule, such as acetylene, and is generally represented byC_(n)H_(2n−3)— (wherein n is a positive integer of two or more). Theterm, “substituted alkynyl” refers to an alkynyl having its Hsubstituted with the following substituent. Specific examples mayinclude C2-C3 alkynyl, C2-C4 alkynyl, C2-C5 alkynyl, C2-C6 alkynyl,C2-C7 alkynyl, C2-C8 alkynyl, C2-C9 alkynyl, C2-C10 alkynyl, C2-C11alkynyl, C2-C12 alkynyl, C2-C3 substituted alkynyl, C2-C4 substitutedalkynyl, C2-C5 substituted alkynyl, C2-C6 substituted alkynyl, C2-C7substituted alkynyl, C2-C8 substituted alkynyl, C2-C9 substitutedalkynyl, C2-C10 substituted alkynyl, C2-C11 substituted alkynyl, orC2-C12 substituted alkynyl. For example, C2-C10 alkynyl refers to astraight chain or branched alkynyl having 2-10 carbon atoms, and isrepresented by ethynyl (CH″C—), 1-propynyl (CH₃C″C—) and the like. Also,for example, the C2-C10 substituted alkynyl refers to a C2-C10 alkynylhaving its one or more hydrogen atoms substituted with a substituent.

As used herein, the term, “alkoxy” refers to a monovalent group having ahydrogen atom removed from the hydroxy group of an alcohol, and isgenerally represented by C_(n)H_(2n+1)O— (wherein n is a positiveinteger of one or more). The term, “substituted alkoxy” refers to analkoxy having its H substituted with the following substituent. Specificexamples of these may include C1-C2 alkoxy, C1-C3 alkoxy, C1-C4 alkoxy,C1-C5 alkoxy, C1-C6 alkoxy, C1-C7 alkoxy, C1-C8 alkoxy, C1-C9 alkoxy,C1-C10 alkoxy, C1-C11 alkoxy, C1-C12 alkoxy, C1-C2 substituted alkoxy,C1-C3 substituted alkoxy, C1-C4 substituted alkoxy, C1-C5 substitutedalkoxy, C1-C6 substituted alkoxy, C1-C7 substituted alkoxy, C1-C8substituted alkoxy, C1-C9 substituted alkoxy, C1-C10 substituted alkoxy,C1-C11 substituted alkoxy, or C1-C12 substituted alkoxy. For example,the C1-C10 alkoxy refers to a straight chain or branched alkoxy having1-10 carbon atoms, and is represented by methoxy (CH₃O—), ethoxy(C₂H₅O—), n-propoxy (CH₃CH₂CH₂O—), and the like.

As used herein, the term “carbocyclic group” refers to a cyclic groupcontaining only carbons, other than the aforementioned “cycloalkyl”,“substituted cycloalkyl”, “cycloalkenyl”, “substituted cycloalkenyl”.The carbocyclic group may be aromatic or non-aromatic, and monocyclic orpolycyclic. The term “substituted carbocyclic group” refers to acarbocyclic group having the H of the carbocyclic group substituted withthe following substituent. Specific examples may include C3-C4carbocyclic group, C3-C5 carbocyclic group, C3-C6 carbocyclic group,C3-C7 carbocyclic group, C3-C8 carbocyclic group, C3-C9 carbocyclicgroup, C3-C10 carbocyclic group, C3-C11 carbocyclic group, C3-C12carbocyclic group, C3-C4 substituted carbocyclic group, C3-C5substituted carbocyclic group, C3-C6 substituted carbocyclic group,C3-C7 substituted carbocyclic group, C3-C8 substituted carbocyclicgroup, C3-C9 substituted carbocyclic group, C3-C10 substitutedcarbocyclic group, C3-C11 substituted carbocyclic group, or C3-C12.substituted carbocyclic group. Also, the carbocyclic group may be C4-C7carbocyclic group, or C4-C7 substituted carbocyclic group. Thecarbocyclic group is represented by a phenyl group having one hydrogenatom deleted. The deletion position of the hydrogen may be anychemically available position, and be on the aromatic ring ornon-aromatic ring.

As used herein, the term, “heterocyclic group” refers to a cyclic grouphaving carbon and a heteroatom. The heteroatom is selected from thegroup consisting of O, S and N, and maybe the same or different. Oneheteroatom, or two or more heteroatoms may be present in theheterocyclic group. The heterocyclic group may be aromatic ornon-aromatic, and monocyclic or polycyclic. The term, “substitutedheterocyclic group” refers to a heterocyclic group having the H of theheterocyclic group substituted with the following substituent. Specificexamples may include those having one or more carbon atoms of thefollowing groups substituted with one or more heteroatoms: C3-C4carbocyclic group, C3-C5 carbocyclic group, C3-C6 carbocyclic group,C3-C7 carbocyclic group, C3-C8 carbocyclic group, C3-C9 carbocyclicgroup, C3-C10 carbocyclic group, C3-C11 carbocyclic group, C3-C12carbocyclic group, C3-C4 substituted carbocyclic group, C3-C5substituted carbocyclic group, C3-C6 substituted carbocyclic group,C3-C7 substituted carbocyclic group, C3-C8 substituted carbocyclicgroup, C3-C9 substituted carbocyclic group, C3-C10 substitutedcarbocyclic group, C3-C11 substituted carbocyclic group, or C3-C12substituted carbocyclic group. Also, the heterocyclic group may be thosehaving one or more carbon atoms of the C4-C7 carbocyclic group or C4-C7substituted carbocyclic group substituted with heteroatoms. Theheterocyclic group is represented by thienyl group, pyrrolyl group,furyl group, imidazolyl group, pyridyl group, and the like. The deletionposition of the hydrogen may be any chemically available position, andbe on the aromatic ring or non-aromatic ring.

As used herein, the term, “carbocyclic group or heterocyclic group” maybe substituted with a divalent substituent, in addition to a monovalentsubstituent, as defined below. Such divalent substitution may be an oxosubstitution (═O) or a thioxo substitution (═S).

As used herein, the term, “halogen” refers to a monovalent group of theGroup 7B element in the Periodic Table such as fluorine (F), chlorine(Cl), bromine (Br) iodine (I), and the like.

As used herein, the term, “hydroxy” refers to a group represented by—OH. The term, “substituted hydroxy” refers to a hydroxy having the H ofthe hydroxy substituted with a substituent as defined below.

As used herein, the term, “thiol” refers to a group (mercapto group)having the oxygen atom of the hydroxy group substituted sulfur atom, andis represented by —SH. The term, “substituted thiol” is a group havingthe H of the mercapto substituted with such a substituent as definedbelow.

As used herein, the term, “cyano” refers to a group represented by —CN.The term, “nitro” is a group represented by —NO₂. The term, “amino” is agroup represented by —NH₂. The term, “substituted amino” is a grouphaving the H of the amino substituted with such a substituent as definedbelow.

As used herein, the term, “carboxy” refers to a group represented by—COOH. The term, “substituted carboxy” is a group having the H of thecarboxy substituted with such a substituent as defined below.

As used herein, the term, “thiocarboxy” refers to a group having theoxygen atom of the carboxy group substituted with sulfur atom, and isrepresented by —C(═S)OH, —C(═O)SH, or —CSSH. The term, “substitutedthiocarboxy” refers to a group having the H of the thiocarboxysubstituted with such a substituent as defined below.

As used herein, the term, “acyl” refers to a monovalent group having theOH removed from the carboxylic acid. Representative examples of the acylgroups include acetyl (CH₃CO—), benzoyl (C₆H₅CO—), and the like. Theterm, “substituted acyl” refers to a group having the hydrogen of theacyl substituted with such a substituent as defined below.

As used herein, the term, “amide” refers to a group having the hydrogenof ammonia substituted with an acidic group (acyl group), and ispreferably represented by —CONH₂ The term, “substituted amide” refers toan amide that is substituted.

As used herein, the term, “carbonyl” collectively refers a group having—(C═O)— that is a characteristic group of aldehyde or ketone. The term,“substituted carbonyl” means a carbonyl group substituted with such asubstituent as defined below.

As used herein, the term, “thiocarbonyl” refers to a group having theoxygen atom of the carbonyl substituted with sulfur atom, and contains acharacteristic group —(C═S)—. The term, “substituted thiocarbonyl” meansa thiocarbonyl substituted with such a substituent as selected below.

As used herein, the term “sulfonyl” is collectively refers to a grouphaving a characteristic group —SO₂—. The term, “substituted sulfonyl”means a sulfonyl substituted with such a substituent as selected below.

As used herein, the term “sulfinyl” is collectively refers to a grouphaving a characteristic group —SO—. The term, “substituted sulfinyl”means a sulfinyl substituted with such a substituent as selected below.

As used herein, the term, “alkylthio” refers to a group having an alkylgroup bonded to a sulfur atom, and is generally represented by —S—R(wherein R is a group having one hydrogen atom removed from the alkyl).

As used herein, the term, “arylthio” refers to a group having an arylgroup bonded to a sulfur atom, and is generally represented by —S'R(wherein R is a group having one hydrogen atom removed from the aryl).

As used herein, the term, “aryl” refers to a group generated by theelimination of one hydrogen atom bonded to the aromatic hydrocarbonring, and is included in a carbocyclic group.

As used herein, the term, “substitution” refers to substituting anorganic group or one or more hydrogen atoms in the substituent of anorganic compound with other atom or atomic group, unless otherwiseindicated. One hydrogen atom may be removed to substitute with amonovalent substituent, or two hydrogen atoms may be removed tosubstitute with a divalent substituent. As used herein, the substituentis defined as follows.

When a substituent R is substituted, R is represented by R^(A)—(R^(B))_(n), wherein R^(A) is a (n+1) group having n hydrogen atomsremoved from R;

R^(B) may be selected from the group consisting of alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, carbocyclic group, substituted carbocyclicgroup, heterocyclic group, substituted heterocyclic group, halogen,hydroxy, substituted hydroxy, thiol, substituted thiol, cyano, nitro,amino, substituted amino, carboxy, substituted carboxy, acyl,substituted acyl, thiocarboxy, substituted thiocarboxy, amide,substituted amide, substituted carbonyl, substituted thiocarbonyl,substituted sulfonyl, and substituted sulfinyl.

When R^(B) is substituted,

R^(B) is represented by R^(C)—(R^(D))_(n), wherein R^(C) is a (n+1)group having n hydrogen atoms removed from R^(B);

R^(D) may be selected from the group consisting of alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, carbocyclic group, substituted carbocyclicgroup, heterocyclic group, substituted heterocyclic group, halogen,hydroxy, substituted hydroxy, thiol, substituted thiol, cyano, nitro,amino, substituted amino, carboxy, substituted carboxy, acyl,substituted acyl, thiocarboxy, substituted thiocarboxy, amide,substituted amide, substituted carbonyl, substituted thiocarbonyl,substituted sulfonyl, and substituted sulfinyl.

When R^(D) is substituted,

R^(D) is represented by R^(E)—(R^(F))_(n), wherein R^(E) is a (n+1)group having n hydrogen atoms removed from R^(D);

R^(F) may be selected from the group consisting of alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, carbocyclic group, substituted carbocyclicgroup, heterocyclic group, substituted heterocyclic group, halogen,hydroxy, substituted hydroxy, thiol, substituted thiol, cyano, nitro,amino, substituted amino, carboxy, substituted carboxy, acyl,substituted acyl, thiocarboxy, substituted thiocarboxy, amide,substituted amide, substituted carbonyl, substituted thiocarbonyl,substituted sulfonyl, and substituted sulfinyl.

When R^(F) is substituted,

R^(F) is represented by R^(G)—(R^(H))_(n), wherein R^(G) is a (n+1)group having n hydrogen atoms removed from R^(F);

R^(1H) or R^(2H) may be selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, carbocyclic group,substituted carbocyclic group, heterocyclic group, substitutedheterocyclic group, halogen, hydroxy, substituted hydroxy, thiol,substituted thiol, cyano, nitro, amino, substituted amino, carboxy,substituted carboxy, acyl, substituted acyl, thiocarboxy, substitutedthiocarboxy, amide, substituted amide, substituted carbonyl, substitutedthiocarbonyl, substituted sulfonyl, and substituted sulfinyl.

When R^(H) is substituted, it may be substituted in the same manner asthe substitution of R^(F), and further substituent may be substituted inthe same manner.

In addition, the number n of the above-discussed substituents may ofcourse be a positive integer, and may be the same or different, and mayeach independently be selected. When n is 2 or more, each substituentthat is represented by ( )n may be the same or different.

As used herein, C1, C2, . . . Cm represents the number of carbons, andC1 is used to represent a substituent having one carbon.

As used herein, the term, “optical isomer” refers to a pair of compoundsor one of such compounds whose crystals or molecular structures are in amirror image relationship, and cannot be overlapped. They are one formof steric isomers, and have the same properties except optical activity.The optical isomers as used herein are preferably those which are notnaturally present in the samples.

(Sample Analysis)

As used herein, the term, “sample” may be available from any sources.These sources include those which use directly or indirectly (via acertain treatment) the whole or portions (for example, organs, tissues,cells, or the like) of organisms (for example, plants).

As used herein, the term, “reverse phase liquid chromatography” refersto a liquid chromatography using the stationary phase having a smallerpolarity than the mobile phase that is reverse to an ordinarychromatography. Normally, in a reverse phase liquid chromatography,those molecules having less hydrophobicity are sequentially eluted.Preferably, a reverse phase liquid chromatography column may be a C18column. As used herein, the term, “C18 reverse phase column(chromatography) ” refers to a reverse phase liquid chromatographycolumn packed with a filler having a carrier (for example, a silica gelcarrier) chemically bonded to a stationary phase (octadecylsilyl, etc.)having a carbon number of 18. The term, “eluent” or “mobile phase”refers to a liquid used for elution in chromatography.

As used herein, “the calculation” from the chromatogram can beaccomplished by extrapolating the actual values, using the relationshipbetween the amounts of the substances used for the measurement standardsand the absorbents, and the relationship between the substances to bemeasured and their standards.

As used herein, the term, “methanol extraction” refers to anorganochemical extraction using methanol as the solvent. Preferably, themethanol extraction may be an extraction using methanol: water=80%:20%(v/v).

As used herein, the term, “etyl acetate extraction” refers to anorganochemical extraction using a solvent comprising etyl acetate as asolvent system. Preferably, the etyl acetate extraction may be anextraction using about 100% ethyl acetate.

As used herein, the term “WRKY family”, “WRKY superfamily” or “WRKY-typetranscription factor” refers to a group of transcription factors withthe WRKY region, which comprises a specific zinc finger motif forplants. The WRKY region is characterized by the consensus amino acidsequence, WRKYCQK (for example, see Trends Plant Sci 2000May;5(5):199-206). Among the WRKY family, there are a number of factorsthat have immediate early type, transiently activated stress responsiveproperties. The WRKY family includes TIZZ and WIZZ. Further, amino acidsas referres to herein may be referred to as either the three-lettersymbol known in the art or the one-letter symbol recommended byIUPAC-IUB Biochemical Nomenclature Commission in general. Additionally,nucleotides may be referred to in the one-letter codes commonlyrecognized.

As used herein, the term “WIZZ (wound-induced leucine zipper zincfinger)” is stress responsive gene (see Hara et al., Mol. Gen. Genet.(2000), 263:30-37) wherein the stress is immediate early type,transiently activated stress (e.g., wound), and belongs to the WRKYfamily. Among the families, it belongs to the group II subfamily. Thisgroup II subfamily includes a WRKY region and a leucine zipper region inthe N-terminus (Trends Plant Sci May 2000;5(5):199-206).

As used herein, the term “TIZZ” is a gene similar to “WIZZ” which hasbeen found in tobacco, and is a wound responsive gene which is animmediate early type, transiently activated (see Yoda et al., Mol.Genet. Gennomics. (2002), 267:152-161), and belongs to the same WRKYgroup II subfamily as WIZZ. This group II subfamily includes a WRKYregion and a leucine zipper region in the N-terminus (Trends Plant SciMay 2000;5(5):199-206).

As used herein, the term “rapid response” refers to a phenomenon thatupon receiving a stimulus, the response to the stimulus occursimmediately (e.g., within one hour, preferably within 30 minutes, morepreferably within 15 minutes) in organisms. In the rapid response, arapid activation of a gene expression including transcription,translation and post-translation modification is involved. As usedherein, “gene” refers to an agent defining a genetic trait. A gene istypically arranged in a given sequence on a chromosome. A gene includesa structural gene which defines the primary structure of protein and aregulatory gene which encodes a protein which regulates the expressionof the structural gene. As used herein, “gene” may refer to“polynucleotide”, “oligonucleotide”, “nucleic acid”, and “nucleic acidmolecule” and/or “protein”, “polypeptide”, “oligopeptide” and “peptide”.

As used-herein, the term “expression” of a gene, a polynucleotide, apolypeptide, or the like, indicates that the gene or the like isaffected by a predetermined action in vivo to be changed into anotherform. Preferably, the term expression indicates that genes,polynucleotides, or the like are transcribed and translated intopolypeptides. In one embodiment of the present invention, genes maybetranscribed into mRNA. More preferably, these polypeptides may havepost-translational processing modifications. As used herein, the term“regulation” includes, but is not limited to, enhancement, reduction,induction, eliminating, delaying, accelerating of the gene expression,and the like.

As used herein, the term “screening” refers to selection of a target,such as an organism, a substance, or the like, a given specific propertyof interest from a population containing a number of elements using aspecific operation/evaluation method. Screening may be performed using asystem in vitro, in vivo, or the like (a system using a real substance)or alternatively a system in silico (a system using a computer). It isunderstood that within the invention, compounds obtained by screeningwould also be encompassed, as long as the compounds have at least oneactivity of those of the present invention.

Therefore, it is contemplated that the present invention provides drugswhich are produced by computer modeling based on the present disclosure.

In other embodiments, the present invention includes compounds obtainedby a quantitative structure activity relationship (QSAR) computermodeling technique as a tool for screening for effectiveness of theregulatory activity of the compound according to the present invention.Here, the computer technique includes some substrate templates preparedby a computer, pharmacophores, production of homologous models of theactive site of the present invention, and the like. In general, a methodfor modeling an ordinary characteristic group of a substance capable ofinteracting with a given substance from data obtained in vitro can becarried out using a CATALYST™ pharmacophore method (Ekins et al.,Pharmacogenetics, 9:477-489, 1999; Ekins et al., J. Pharmacol. & Exp.Ther., 288:21-29, 1999; Ekins et al., J. Pharmacol. & Exp. Ther.,290:429-438, 1999; Ekins et al. , J. Pharmacol. & Exp. Ther.,291:424-433, 1999) and comparative molecular field analysis; CoMFA)(Jones et al., Drug Metabolism & Disposition, 24:1-6, 1996), and thelike. In the present invention, the computer modeling may be carried outusing molecular modeling software (e.g., CATALYST™ version 4 (MolecularSimulations, Inc., San Diego, Calif.), etc.).

Fitting of a compound to an active site can be carried out using anycomputer modeling technique known in the art. Visual inspection andmanual operation of a compound to an active site can be carried outusing a program, such as QUANTA (Molecular Simulations, Burlington,Mass., 1992) SYBYL (Molecular Modeling Software, Tripos Associates,Inc., St. Louis, Mo., 1992), AMBER (Weiner et al., J. Am. Chem. Soc.,106:765-784, 1984), CHARMM (Brooks et al., J. Comp. Chem., 4:187-217,1983), or the like. In addition, energy minimization can be carried outusing a standard force field, such as CHARMM, AMBER, or the like. Othermore specialized computer modelings include GRID (Goodford et al., J.Med. Chem., 28:849-857, 1985), MCSS (Miranker and Karplus, Function andGenetics, 11:29-34, 1991), AUTODOCK (Goodsell and Olsen, Proteins:Structure, Function and Genetics, 8:195-202, 1990), DOCK (Kuntz et al.,J. Mol. Biol. , 161:269-288, (1982)), and the like. Additionalstructures of compounds can be newly constructed with blank activesites, active sites of known low molecular weight compounds, or thelike, using a computer program, such as LUDI (Bohm, J. Comp. Aid. Molec.Design, 6:61-78, 1992), LEGEND (Nishibata and Itai, Tetrahedron,47:8985, 1991), LeapFrog (Tripos Associates, St. Louis, Mo.), or thelike. Such computer modelings are well known in the art and commonlyused. Those skilled in the art can appropriately design compounds withinthe scope of the present invention in accordance with the disclosures ofthe present specification.

WIZZ is a disease responsive WRKY transcription factor whosetranscription product is extremely rapidly accumulated when wound (Mol.Gen. Genet, 2000:263.30-37) and when infected with a pathogen (Mol.Genet. Genomic. 2002 267.154-161). It is indicated that some types ofWRKY type transcription factor are activated by infection with apathogen, the expression of pathogen-related proteins as a target geneis induced, are therefore involved in the defense response against theinfection of the pathogen (Genes & Development 2002, 16, 1139-1149). Assuch, it is demonstrated that in plants, activation of the WRKY typetranscription factor triggers physiological reactions in vivo.Therefore, if WIZZ is activated by the compound of the invention, thenthe expression of downstream defense-related gene(s) as targets areinduced, and the resistant reaction against disease/wound stress isinduced.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention provides a compound having the followingstructure:

wherein, in the formula:

X is selected from the group consisting of hydroxy, substituted hydroxy,halogen, thiol, or substituted thiol. Preferably, X may be hydroxy orsubstituted hydroxy. More preferably, it may be hydroxy oralkyl-substituted hydroxy. Even more preferably, it may be hydroxy.

One of Y¹ and Y² may be hydrogen or alkyl, and the other may be Z-W,wherein Z may be a single bond, or a divalent group having alkane orsubstituted alkane having two hydrogen atoms removed, and W may behydroxy, substituted hydroxy, aldehyde, carboxyl, or substitutedcarboxyl, but both of Y¹ and Y² may be Z-W, wherein Z may be a singlebond, or a divalent group having alkane or substituted alkane having twohydrogen atoms removed, and W may be hydroxy, aldehyde, carboxyl, orsubstituted carboxyl. Preferably, one of Y¹ and Y² may be hydrogen, andthe other may be methylol, substituted methylol, C1-aldehyde,C1-carboxyl, or C1-substituted carboxyl. More preferably, one of Y¹ andY² may be hydrogen, and the other may be methylol, alkyl-substitutedmethylol, C1-aldehyde, C1-carboxyl, or C1-alkyl-substituted carboxyl.Even more preferably, one of Y¹ and Y² may be hydrogen, and the othermay be methylol, methyl, or ethyl-substituted methylol, C1-aldehyde,C1-carboxyl, or C1-methyl or ethyl-substituted carboxyl.

R¹˜R²⁴ is independently selected from the group consisting of hydrogen,alkyl, substituted alkyl, cyclo alkyl, substituted cyclo alkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, carbocyclic group,substituted carbocyclic group, heterocyclic group, substitutedheterocyclic group, halogen, hydroxy, substituted hydroxy, thiol,substituted thiol, cyano, nitro, amino, substituted amino, carboxy,substituted carboxy, acyl, substituted acyl, thiocarboxy, substitutedthiocarboxy, amide, substituted amide, substituted carbonyl, substitutedthiocarbonyl, substituted sulfonyl, and substituted sulfinyl.Preferably, R¹-R²⁴ may independently be selected from the groupconsisting of hydrogen, alkyl, and substituted alkyl. More preferably,it may be independently selected from the group consisting of hydrogen,and C1-C6 alkyl. All of R¹-R²⁴ may have substituents other thanhydrogen, but preferably, have at least one hydrogen, more preferably,two hydrogens, three hydrogens, four hydrogens, five hydrogens, sixhydrogens, seven hydrogens, eight hydrogens, nine hydrogens, tenhydrogens, eleven hydrogens, twelve hydrogens, thirteen hydrogens,fourteen hydrogens, fifteen hydrogens, sixteen hydrogens, seventeenhydrogens, eighteen hydrogens, nineteen hydrogens, twenty hydrogens,twenty one hydrogens, twenty two hydrogens, twenty three hydrogens. Thepresence of more hydrogens in the R¹-R²⁴ substituent may be preferable,because a larger substituent may interfere with the effects of thepresent invention. Therefore, preferred substituents other than hydrogenmay include C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2alkyl, methyl, and the like. However, a larger substituent may bepreferred because it may enhance the effects of the invention. Morepreferably, all of R¹-R²⁴ may be hydrogens.

In one preferred embodiment, the present invention provides a compoundhaving the following structural formula:

(WAF-1)

This compound is a Labdan-type diterpene compound that is(11E,13E)-labda-11,13-diene-8″,15-diol.

The compounds of the present invention can be synthesized by using anyof known techniques in the art. Such synthetic examples are illustratedbelow, but the synthesis methods of the present invention are notlimited to these examples.

In one aspect, the synthesis method of the present invention comprisesfollowing steps:

1) reacting a compound:

wherein, in the formula:

R⁵-R²⁴ are independently selected from the group consisting of hydrogen,alkyl, and substituted alkyl, and the same as R¹-R²⁴ for WAF, with analkyl lithium to provide an intermediate 2;

wherein the intermediate 1 may be dissolved in anhydrous Et₂O (diethylether), and the alkyl lithium may be methyl lithium. The alkyl lithiummay be dissolved in the Et₂O. The alkyl lithium may be added dropwisewhile stirring as cooling on ice under a nitrogen atmosphere. After thereaction, for example, the reactant may be extracted into the Et₂O layerover five minutes under an acidic condition (e.g., 10% H₂SO₄). Theproduct maybe eluted into a suitable solvent (for example, using asilica gel column chromatography with hexane-Et₂O). The product may beidentified as having the desirable structure by measuring the propertieswith NMR and MS.

2) mixing and reacting the product obtained in 1) withm-chloroperbenzoic acid and then with a 10% potassium hydroxide inmethanol to provide an intermediate 4;

wherein the product may be an acetyl intermediate before being convertedinto the intermediate 4. The mixing can be accomplished while stirringon ice under a nitrogen stream. The residue may be dissolved into asolution of an alkali (for example, 10% KOH) in methanol. The reactionmixture may be extracted into Et₂O. The extract may be washed with asaturated aqueous NaHCO₃ solution, water, and a saturated brine.Optionally, it may be dried with Na₂SO₄. It may be eluted with a flashchromatography (hexane-AcOEt(3:1)).

3) reacting the product obtained in 2) with N-methylmorphorine N-oxideto provide an intermediate 5;

wherein the N-methylmorphorine N-oxide may be suspended in anhydrousCH₂Cl₂ with 4 Å molecular sieve. These reactions may be carried outwhile stirring on ice under a nitrogen stream. To this, tetrapropylammonium peruthenate may be added and reacted with. To the reactionmixture, Et₂O may be added, stirred, and filtered with silica gel. Thefiltrate may be concentrated, subjected to a flash chromatography, andthe product may be eluted into the hexane-Et₂O elution portion.

(4) adding a compound

wherein, one of V¹ and V² is hydrogen or alkyl, and the other is Z-V,and wherein Z is (CH2)n-C(═O)—O—, V is alkyl, n is an integer of 0 ormore, and R is alkyl (wherein R may preferably be a lower alkyl, andmore preferably methyl), to said intermediate 5 obtained in the step (3)in an organic solvent in the presence of NaNH₂ to provide anintermediate (6):

wherein NaNH₂ may be suspended in anhydrous THF, followed by theaddition of the compound 28 while stirring on ice under a nitrogenstream. The reaction mixture may be cooled at −78′, to which theintermediate 5 maybe added. The product may be extracted into Et₂O. Theproduct may suitably be dried with Na₂SO₄. This may be isolated with aflash chromatography using hexane-AcOEt. The optical isomers may furtherbe eluted with high performance liquid chromatography as usinghexane-AcOEt and the like as the solvent;

(5) adding diisobutyl aluminum hydride in an organic solvent to saidintermediate (6) obtained in the step (4) to provide

wherein, X is selected from the group consisting of hydroxy, substitutedhydroxy, halogen, thiol, and substituted thiol;

one of U¹ and U² is hydrogen or alkyl, and the other is Z-U, wherein Zis a single bond, or a divalent group having alkane or substitutedalkane having two hydrogen atoms removed, and U is hydroxy; and

R¹-R²⁴ are independently selected from the group of hydrogen, alkyl, andsubstituted alkyl;

wherein the diisobutyl aluminum hydride may be dissolved in CH₂Cl₂(preferably, under nitrogen stream), and thereafter returned to roomtemperature. The product may be added with AcOEt, CH₂Cl₂, potassiumsodium tartrate and the like, and extracted with AcOEt. It may suitablybe dried, and eluted with a flash chromatography usinghexane-AcOEt(1:1).

Optionally, the method may comprise a further oxidation or substitutionstep where Y¹ is other than hydroxy.

In the above-described synthesis method, the substituents may beconverted according to the following techniques where Y¹ is other thanhydroxy.

Conversion from hydroxy into aldehyde: [MnO₂, heptane, 25° C.]: N. L.Wendler et al., J. Am. Chem. Soc., Vol. 73, 719 (1951); [CrO₃, pyridine,25° C.]: J. R. Holum, J. Org. Chem., Vol. 26, 4814(1961).

Conversion from hydroxy into substituted methylol: [NaH, MeI, THF,25°C.]: C. A. Brown et al. , Synthesis, 1974, 434.

Conversion from hydroxy into carboxyl: [NiO₂, aqueous 1N NaOH solution,50° C.]: K. Nakagawa et al., J. Org. Chem., Vol. 27, 1597(1962).

Examples of the starting materials in the above-described step 1)include a compound (sclareolide) having the structural formula:

Sclareolide is commercially available, or may be synthesized as follows:

Therefore,

can readily be synthesized by those skilled in the art in view of thesynthesis example of the sclareolide.

The reaction schemes when the above-described representative examplesare illustrated below:

(Pharmaceutical Compositions, Agrichemical Compositions)

In another aspect, the present invention provides a compositioncomprising the compound of the present invention. These compositions maybe pharmaceutical compositions, or agricultural (pesticide)compositions.

When the factor or compound is formulated into a pesticide orpharmaceutical composition, such a composition may contain anagriculturally or pharmaceutically acceptable carrier. These carriersinclude any materials known in the art.

These appropriate agriculturally or pharmaceutically acceptable factorsinclude, but are not limited to the following: antioxidants,preservatives, colorants, flavoring agents, diluents, emulsifiers,suspending agents, solvents, fillers, extenders, buffers, deliveryvehicles, excipients, and/or agricultural or pharmaceutical adjuvants.Typically, the pesticide or pharmaceutical composition of the presentinvention may be administered as a composition comprising theLabdan-type diterpenoid compound of the present invention together withone or more physiologically acceptable carriers, excipients or diluents.For example, an appropriate vehicle for the pharmaceutical compositionmay be water for injection, a physiological solution, or an artificialcerebral fluid, which may be supplemented with other materials that arecommon in a composition for parental administration. An appropriatevehicle for the pesticide composition may water for pesticideadministration.

Illustrative appropriate carriers include neutral buffered saline, orsaline mixed with serum albumin. Preferably, the product is formulatedas a freeze-dried agent using a suitable excipient (for example,sucrose). Optionally, it may contain other standard carriers, diluents,and excipients. Other illustrative composition contains a Tris buffer ofpH 7.0-8.5, or an acetic acid buffer of pH 4.0-5.5, and may furthercontain sorbitol, or its suitable alternative. The pH of the solutionshould be selected based on the relative solubility of the factor of thepresent invention.

The solvent for the composition may be either aqueous or non-aqueous.Further, its vehicle may contain other formulations for modifying orretaining the pH, osmolarity, viscosity, clarity, color, sterility,stability, isotonicity, disintegration rate, or odor. Similarly, thecomposition of the present invention may contain other formulations formodifying or retaining a rate of the active ingredients, or facilitatingthe absorption or permeation of the active ingredients.

When the composition of the present invention is formulated as apharmaceutical composition, it may be parenterally administered.Alternatively, the composition may be intravenously or subcutaneouslyadministered. When the pharmaceutical composition used in the presentinvention is systemically administered, it may be in the form of anorally acceptable aqueous solution that does not contain any pyogenicsubstance. The preparation of such a pharmaceutically acceptable proteinsolution is within the skill of the art, provided that attention begiven to pH, isotonicity, stability, and the like.

When the composition of the present invention is formulated as apharmaceutical composition, it may be prepared for storage in the formof a freeze-dried cake or aqueous solution by optionally mixing theselected composition having a desirable degree of purity with aphysiologically acceptable carrier, excipient, or stabilizer (See,Pharmacopoeia of Japan; Remington's Pharmaceutical Sciences, 18thEdition, A. R. Gennaro, ed., Mack Publishing Company, 1990, etc.).

When the composition of the present invention is formulated as apesticide composition, it may optionally contain an agriculturallyacceptable carrier, excipient, or stabilizer, or the like.

The acceptable carriers, excipients, or stabilizers are non-toxic to therecipient, and preferably inactive to the dosage and concentration to beused, and include the following: phosphates, citrates, or other organicacids; antioxidants (such as ascorbic acid); low molecular weightpolypeptides; proteins (such as serum albumin, gelatin, orimmunoglobulin); hydrophilic polymers (such as polyvinyl pyrrolidone);amino acids (such as glycine, glutamine, arginine, or lysine);monosaccharides, disaccharides, and other carbohydrates (includingglucose, mannose, or dextrin); chelating agents (such as EDTA); sugaralcohol (such as mannitol, or sorbitol); salt-forming counterions (suchas sodium); and/or nonionic surfactants (such as Tween, Pluronic, orpolyethylene glycol (PEG)).

When the composition of the present invention is used as a pesticidecomposition, the following pesticide active ingredients may concurrentlybe contained:

(herbicides) pyrazonate, daimuron, bromobutide, mefenacet, MCP, MCPB,triclopyr, naproanilide, CNP, chlomethoxynil, bifenox, MCC,pyributicarb, DCPA, napropamide, diphenamid, propyzamide, asulam, DCMU,linuron, methyldymron, tebuthiuron, bensulfuronmethyl, simazine,atrazine, simetryn, ametryn, prometryn, dimethametryn, metribuzin,bentazone, oxadiazon, pyrazonate, benzofenap, glyphosate, bilanafos,alloxydim, imazosulfuron, azimsulfuron, pyrazosulfuron, cinosulfuron;

(insecticides/acaricides) diazinon, fenthion, isoxathion,pyridaphenthion, fenitrothion, dimethoate, PMP, dimethylvinphos,acephate, DEP, NAC, MTMC, MIPC, PHC, MPMC, XMC, BPMC, bendiocarb,pirimicarb, methomyl, oxamyl, thiodicarb, cypermethrin, cartaphydrochloride, thiocyclam, bensultap, pyriproxyfen, phenoxycarb,methoprene, diflubenzuron, teflubenzuron, chlorfluazuron, buprofezin,hexythiazox, pyridaben, clofentezine, nitenpyram;

(bactericides) probenazole, isoprothiolane, pyroquilon, flutolanil,metominostrobin, ziram, thiram, captan, TPN, phthalide,tolclofos-methyl, fosetyl, thiophanate methyl, benomyl, carbendazole,thiabendazole, diethofencarb, iprodione, vinclozolin, procymidone,fluoroimide, oxycarboxin, mepronil, flutolanil, pencycuron, metalaxyl,oxadixyl, triadimefon, hexaconazole, triforine, blasticidin-S,kasugamycin, polyoxin, validamycin-A, mildiomycin, PCNB,hydroxyisoxazole, dazomet, dimethirimol, diclomezine, triazine,ferimzone, tricyclazole, oxolinic acid, and the like, and preferablystrobilurin-based compounds, such as metominostrobin and the like.

When a composition of the present invention is used as an agriculturalchemical, the composition may be mixed with an acaricide (e.g.,chlorobenzilate, etc.), a plant growth regulator (e.g., paclobutrazol,etc.), anematocide (e.g., benomyl, etc.), a synergist (e.g., piperonylbutoxide, etc.), an attractant (e.g., eugenol, etc.), a repellent (e.g.,creosote, etc.), a pigment (e.g., food blue No. 1, etc.), a fertilizer(e.g., urea, etc.), or the like.

When the composition of the present invention is used as apharmaceutical composition, the composition further contains thefollowing medicinal ingredients:

central nerve system drugs (e.g., general anesthetics,sedative-hypnotics, anxiolytics, antiepileptics, anti-inflammatoryagents, stimulants, antihypnotics, antiparkinson agents, antipsychotics,combination cold remedies, and the like);

peripheral nerve agents (e.g., local anesthetics, skeletal musclerelaxants, autonomic nerve agents, antispasmodic agents, and the like);

sensory organ drugs (e.g., ophthalmological agents,otorhinolaryngological agents, antidinics, and the like);

circulatory organ drugs (e.g., cardiotonics, antiarrhythmics, diuretics,antihypertensive agents, vasoconstrictors, vasodilators,antihyperlipemia agents, and the like);

respiratory organ drugs (e.g., respiratory stimulants, antitussives,expectorants, antitussive extpectorants, bronchodilators, collutoriums,and the like);

digestive organ drugs (e.g., stegnotics, antiflatuents, peptic ulceragents, stomachics, antacids, cathartics, enemas, cholagogues, and thelike);

hormone agents (e.g., pituitary gland hormone agents, salivary glandhormone agents, thyroid gland hormone agents, accessory thyroid glandhormone agents, anabolic steroid agents, adrenal gland hormone agents,androgenic hormone agents, estrogen agents, progesterone agents, mixedhormone agents, and the like);

urogenital organ and anal drugs (e.g., urinary organ agents, genitalorgans agents, uterotonics, hemorrhoids agents, and the like);

dermatologic drugs (e.g., dermatologic disinfectants, wound protectingagents, pyogenic diseases agents, analgesics, antipruritics,astringents, antiphlogistics, parasitic skin diseases agents,emollients, hair agents, and the like);

dental and oral agents;

drugs for other organs;

vitamin agents (e.g., vitamin A agents, vitamin D agents, vitamin Bagents, vitamin C agents, vitamin E agents, vitamin K agents, mixedvitamin agents, and the like);

nutritive agents (e.g., calcium agents, inorganic preparations,saccharide agents, protein amino acid preparations, organ preparations,infant preparations, and the like);

blood and body fluid drugs (e.g., blood substitute agents, styptics,anticoagulants, and the like);

dialysis drugs (e.g., kidney dialysis agents, peritoneal dialysisagents, and the like);

other metabolic drugs (e.g., organ disease agents, antidotes, antabuses,arthrifuges, enzyme preparation, diabetic agents, and others);

cell activating agents (e.g., chlorophyll preparations, pigment agents,and the like);

tumor agents (e.g., alkylation agents, antimetabolites, antineoplasticantibiotic preparations, antineoplastic plant extract preparations, andthe like);

radiopharmaceuticals;

allergy drugs (e.g., antihistamine agents, irritation therapy agents,non-specific immunogen preparations, and other allergy drugs, crudedrugs and drugs based on Chinese medicine, crude drugs, Chinese medicinepreparations, and other preparations based on crude drug and Chinesemedicine formulations);

antibiotic preparations (e.g., acting for gram-positive bacteria,gram-negative bacteria, gram-positive mycoplasmas, gram-negativemycoplasmas, gram-positive rickettsia, gram-negative rickettsia,acid-fast bacteria, molds, and the like);

chemotherapeutic agents (e.g., sulfa drugs, antitubercular agents,synthetic antimicrobial agents, antiviral agents, and the like);

biological preparations (e.g., vaccines, toxoids, antitoxins, leptospireantisera, blood preparations, biological test preparations, and otherbiological preparations, and antiprotozoal drugs, anthelmintics, and thelike);

dispensing agents (e.g., excipients, ointment bases, solvents, flavors,colorants, and the like);

diagnostic drugs (e.g., contrast media, function testing reagents, andthe like);

sanitation drugs (e.g., preservatives);

xenodiagnostic drugs (e.g., cytologic examination drugs, and the like);

non-categorized drugs which do not aim mainly for therapy; and

narcotics (e.g., opium alkaloid drugs, coca alkaloid preparations,synthetic narcotics, and the like)

Although the following illustrate the detailed description of theapplication of the present invention to plants, which is the preferredaspect of the present invention, it is to be understood that the presentinvention may be applied to other organisms such as animals.

In one embodiment, the composition of the present invention is providedfor imparting stress resistance to a plant or augmenting said stressresistance. Preferably, said stress resistance comprises at least oneresistance selected from the group consisting of wound resistance,insect resistance, disease resistance, and hypersensitivity cell deathresistance.

In certain embodiments, the imparting or augmenting of said stressresistance to an organism in the present invention is accomplished bycontrolling the activity of at least one protein selected from the groupconsisting of wound-induced protein kinases, and salicylic acid-inducedprotein kinases. The wound-induced protein kinases, and salicylicacid-induced protein kinases are known in the art.

In another embodiment, the imparting or augmenting of said stressresistance to an organism in the present invention is accomplished bycontrolling at least one signaling system selected from the groupconsisting of jasmonic acid signaling systems and salicylic acidsignaling systems. The jasmonic acid signaling systems are outlined in“Cell Technology Additional Volume, Plant Cell Technology Series 10, TheSignaling Systems of Plant hormones—Biosynthesis to Physiology—(HirotoyoFukuda, ed.), Chapter 6 (The Physiology, Biosynthesis, and SignalingSystems of Jasmonic Acid, pages 190-198)”, and the like, and thesalicylic acid signaling systems are outlined in “The Plant Cell, Vol.13, 1877-1889, 2001”, and the like.

In another aspect, the present invention provides a method of impartingstress resistance to an organism such as a plant or augmenting saidstress resistance, wherein said method comprises the following steps:

1) applying the compound or composition of the present invention to saidplant. In a preferred embodiment, said compound may be a compound havingsuch a preferred substituent as described above. In a preferredembodiment, said compound may take the form of a composition (such asagricultural composition).

In one preferred embodiment, said stress resistance may be at least oneresistance selected from the group consisting of wound resistance,insect resistance, disease resistance, and hypersensitivity cell deathresistance.

In another preferred embodiment, the imparting or augmenting of saidstress resistance may be accomplished by controlling the activity of atleast one protein selected from the group consisting of wound-inducedprotein kinases, and salicylic acid-induced protein kinases.

In another preferred embodiment, the imparting or augmenting of saidstress resistance may be accomplished by controlling at least onesignaling system selected from the group consisting of jasmonic acidsignaling systems and salicylic acid signaling systems.

In another aspect, the present invention provides a method of producingstress resistant plants. Said method comprises:

1) applying the compound or composition of the present invention to saidplant. In a preferred embodiment, said compound may be a compound havingsuch a preferred substituent as described above. In a preferredembodiment, said compound may take the form of a physiologicallyacceptable composition (such as agricultural composition) The presentinvention also relates to a plant obtained by such a method. The plantcan be obtained by a technique well known in the art. For example, theplant can be obtained by spraying the composition of the presentinvention on a certain plant.

In another aspect, the present invention provides a method of producingstress resistant plant tissues. Said method comprises:

1) applying the compound or composition of the present invention to saidplant tissue. In a preferred embodiment, said compound may be a compoundhaving such a preferred substituent as described above. In a preferredembodiment, said compound may take the form of a physiologicallyacceptable composition (such as agricultural composition) The presentinvention also relates to a plant tissue obtained by such a method. Atechnique for obtaining the plant tissue is well known in the art. Forexample, the plant tissue may be a tissue of the plant structure such ascallus, stem, leaf, flower, seed, and the like. In such a case, it canbe made by isolating the plant tissue, and applying the compound orcomposition of the present invention to the isolated plant tissue.

In another aspect, the present invention provides a method of producingstress resistant plant cells. Said method comprises:

1) applying the compound or composition of the present invention to saidplant cell. In-a preferred embodiment, said compound may be a compoundhaving such a preferred substituent as described above. In a preferredembodiment, said compound may take the form of a physiologicallyacceptable composition (such as agricultural composition) The presentinvention also relates to a plant cell obtained by such a method.

In another aspect, the present invention provides a method of producingstress resistant plant seeds. Said method comprises:

1) applying the compound or composition of the present invention to saidplant; and obtaining said seed from said plant. In a preferredembodiment, said compound may be a compound having such a preferredsubstituent as described above. In a preferred embodiment, said compoundmay take the form of a physiologically acceptable composition (such asagricultural composition). The present invention also relates to a plantseed obtained by such a method. A technique for obtaining the plant seedis well known in the art. For example, if the plant is a floweringplant, the seed can be obtained by fertilizing the plant using anytechnique well known in the art.

In another aspect, the present invention provides a method ofquantifying the compound of the present invention: The methodcomprises 1) providing a sample; 2) adding the predetermined amount ofthe steric isomer of a compound to be quantified to said sample; 3)separating said sample by a reverse phase liquid chromatography; and 4)calculating the amount of said compound from said separated stericisomer. In a preferred embodiment, said compound to be quantified hasthe following structural formula:

said steric isomer has the following structural formula:

Preferably, said sample is extracted with methanol and subsequently withethyl acetate, prior to the separation with said reverse phase liquidchromatography. This is advantageous for the measurement of the presentcompound to be measured, since the methanol extraction is expected tosupress the metabolism and decomposition of the present compound due tothe enzyme reaction, and the ethyl acetate extraction is expected toimprove the extraction efficiency. The ratio of each of these pluralsolvents may be varied depending upon the conditions of the sample.Therefore, the methanol extraction may be an extraction of a lower(preferably, C1-C6) alcohol such as ethanol. Preferably, the methanolextraction may be an extraction using methanol: water=80%:20% (v/v), butother ratios may be used. Preferably, the ethyl acetate extraction maybe an extraction using about 100% ethyl acetate, but those having othersolvents (such as chloroform) may be used.

Preferably, the separation with said reverse phase liquid chromatographycomprises a separation with a C18 reverse chromatography column, andsaid separation comprises a first separation in 80%:20% (v/v)methanol:water, anda separationwith 9:8 (v/v) acetonitrile:water. Theratio of each of these plurality of solvents may be varied dependingupon the conditions of the sample and the kind of compound to bemeasured. Therefore, solvent systems such as water/2-propanol,water/ethanol, and the like can be used.

Preferably, said calculation comprises the correction of the recoveryloss. The recovery loss can be determined by X=Y·100/Z, wherein Y is theactual measurement value, X is the amount at the 100% recoverypercentage, and Z(%) is the actual recovery percentage.

In another aspect, the present invention provides a composition forinducing a rapid accumulation of a WRKY family gene in a plant under acondition requiring the accumulation of a WRKY family gene. Thecomposition comprises the compound of the present invention. Thecomposition may further comprise agriculturally acceptable substances(such as excipients). The composition may also comprise otheragriculturally active agents.

In a preferred embodiment, said compound has the following structuralformula:

In a preferred embodiment, the condition requiring the accumulation ofsaid WRKY family gene may be a condition requiring the rapid response tostress.

Preferably, said plant may be provided with wound resistance, insectresistance, disease resistance, and hypersensitivity cell deathresistance by inducing a rapid accumulation (for example, within 30minutes, more preferably within 15 minutes) of said WRKY family gene.

Preferably, said WRKY family gene may be WIZZ or TIZZ.

In another aspect, the present invention provides a composition forregulating the expression of a WRKY family gene. Said compositioncomprises the compound of the present invention. The composition mayfurther comprise agriculturally acceptable substances (such asexcipients) The composition may also comprise other agriculturallyactive agents.

In another aspect, the present invention provides a method of inducing arapid accumulation of a WRKY family gene in a plant under a conditionrequiring the accumulation of a WRKY family gene. Said method comprisesa) applying to said plant the compound of the present invention.

Preferably, said compound has the following structural formula:

In a preferred embodiment, the condition requiring the accumulation ofsaid WRKY family gene may be a condition requiring the rapid (forexample, within 30 minutes, more preferably within 15 minutes) responseto stress.

In a preferred embodiment, said plant may be provided with woundresistance, insect resistance, disease resistance, and hypersensitivitycell death resistance by inducing a rapid accumulation of said WRKYfamily gene.

In the above method, preferably, said WRKY family gene may be WIZZ orTIZZ. The preferred gene may be varied depending upon the plant varietyto be treated.

Preferably, the compound of the present invention may be appliedimmediately after the accumulation of said WRKY family gene is required.The application techniques include various techniques such as directspraying, and the like.

In another aspect, the present invention provides a composition forregulating the expression of a WRKY family gene. Said compositioncomprises the compound of the present invention. The composition mayfurther comprise agriculturally acceptable substances (such asexcipients) The composition may comprise other agriculturally activeagents.

In a further aspect, the present invention provides a composition forfacilitating the elongating growth or auxetic growth of a plant.

In another aspect, the present invention provides a composition forinhibiting the elongating growth of a plant.

In further aspect, the present invention provides a composition forfacilitating the maturation of a plant.

In further aspect, the present invention provides a composition forregulating the flowering of a plant.

In preferred embodiments, these compositions comprise the compound ofthe present invention. The composition may further compriseagriculturally acceptable substances (such as excipients). Thecomposition may also comprise other agriculturally active agents.

Preferably, the compound of the present invention may facilitate theelongating growth or auxetic growth of the above-described plant,inhibit the elongating growth of the plant, facilitate the maturation ofthe plant, or regulate the flowering of the plant by the rapidaccumulation of an ACO gene, or the rapid accumulation of ethylene. Thetechniques for these include various techniques such as direct spraying,diffusing, and the like.

In another aspect, the present invention provides a method offacilitating the elongating growth or auxetic growth of a plant. Themethod comprises 1) applying the compound of the present invention tothe above-described plant.

In another aspect, the present invention provides a method of inhibitingthe elongating growth of a plant. The method comprises 1) applying thecompound of the present invention to the above-described plant.

In a further aspect, the present invention provides a method offacilitating the maturation of a plant. The method comprises 1) applyingthe compound of the present invention to the above-described plant.

In a further aspect, the present invention provides a method ofcontrolling the flowering of a plant. The method comprises 1) applyingthe compound of the present invention to the above-described plant.

In a preferred embodiment, said compound may be a compound having such apreferred substituent as described above. In a preferred embodiment,said compound may take the form of a physiologically acceptablecomposition (such as agricultural composition). The present inventionalso relates to a plant obtained by such a method. The plant can beobtained by a technique well known in the art. For example, the plantcan be obtained by spraying the composition of the present invention oncertain plant.

In one aspect, the present invention provides a medium for regulatingthe growth (proliferation), differentiation or regeneration of a plant,plant structure or plant tissue, or plant cell. The medium contains thecompound of the present invention. The medium may further containingredients that are employed in other common cell cultures, and planttissue cultures. The medium may also contain agriculturally acceptablesubstances (such as excipients).

The following examples illustrate the present invention, but theseexamples are for the purpose of illustration only. The scope of thepresent invention is not limited by the examples, but by only theclaims.

EXAMPLES Example 1 Isolation and Purification of WIPK Activator inTobacco

(Plant Material)

Tobacco leaf infected with tobacco mosaic virus (TMV) was used as amaterial in this example to conduct isolation of WIPK activator becauseit was known that WIPK was dramatically activated in tobacco leafinfected with TMV.

Superior healthy leaves of two-month-old tobacco (Nicotiana tabacum cv.Samsun—NN) plant bodies were cut and silicon carbide (brand name:Carborundum, Kishida Chemical Co., Ltd., Osaka, Japan) was applied tothe epidermis, against which 10 mM sodium phosphate buffer (pH 7.0)containing TMV was rubbed to inoculate with TMV. To complete theinfection, this healthy leaf was allowed to stand at room temperaturefor 30 minutes and washed with water removing the silicon carbide, afterair drying, was put into a transparent plastic box spread with wetfilter papers, followed by culture at 30° C. for 40 hours, then at 20°C. for 6 hours.

The light condition during culture was continuous irradiation with whitefluorescent light (approximately 6,000 lux). The inoculated leaf, afterculture, was snap-frozen in liquid nitrogen and immediately used asmaterial from which to prify WIPK activator.

(Purification of WIPK Activator)

Plant material was put into a homogenizer (brand name: Polytron,Kinematica, Switzerland) and homogenized in 4 volumes of cold 80% (v/v)acetone (i.e. 4 ml for 1 g material) to a fine powder, and thehomogenate was extracted by standing at 4° C. for two hours. The extractwas filtered with a filter paper (Toyo Roshi Kaisha, Co., Ltd., Japan).The residue after filtration was washed with a small amount of 80% (v/v)acetone and, combinedwiththe above filtrate, and concentrated underreduced pressure at 35° C. to the aqueous phase. The resultant aqueousphase was adjusted with hydrochloric acid to pH 3.0 and extracted withethyl acetate of the same amount three times. The resultant ethylacetate phase was extracted with the same amount of 5% (w/v) sodiumbicarbonate twice, then the surface ethyl acetate phase was dehydratedwith anhydrous sodium sulfate and concentrated under reduced pressure at35° C. to dryness.

The dried substance was dissolved in hexane containing a small amount of10% (v/v) ethyl acetate and the dissolved substance was applied to acolumn (internal diameter 3 cm, length 50 cm) and filled with silica gel(Wakogel C-200, Wako Pure Chemical, Osaka, Japan). A mixed solution ofethyl acetate/hexane was used as the solvent system. A mixed solution at10% (v/v) of ethyl acetate concentration in the mixture of hexane andethyl acetate was applied first and then a mixed solution at 20% (v/v)was then applied, followed by a stepwise increase in increments of 10%.The mixed solution was applied in 900 ml aliqots each time. Theresultant eluted fractions was concentrated under reduced pressure at35° C. to dryness and each dried substance was dissolved in 10 ml ethylacetate, some of which (3, 30, 100 and 300 μl) were subjected to a WIPKinduced activity assay.

The WIPK induced activity assay was conducted as follows: A filter paperwas spread in a glass Petri dish of 3 cm inside diameter, onto which thetest solution for assay was added, organic solvent was removed under theflow of nitrogen gas, and 10 mM Mes-NaOH (pH 5.6) of 1 ml was added, onwhich three leaf disks (diameter 9 mm) stamped out of the tobacco leaveswere placed per Petri dish and cultured at 24° C. In two hours, thedisks were collected and immediately frozen in liquid nitrogen to beused in WIPK activity measurement that was conducted following themethod described in Seo et al. (1999) Plant Cell 11, 289-291. Theoutline is as follows:

Crude protein of 50 μg and anti-WIPK antibody were reacted and themyelin basic protein phosphorylation activity of WIPK in the resultantimmune complex was measured.

WIPK activity was detected in the fractions from 60% (v/v) to 80% (v/v)of ethyl acetate concentration in hexane. Therefore, these fractionswere combined and concentrated under reduced pressure at 35° C. todryness. The resultant dried substance was dissolved in water containinga small amount of 10% (v/v) methanol and the dissolved substance wasadded into a solid phase extraction column cartridge of reverse phasetype (C18 Sep-Pak, Waters, USA). A mixed solution of methanol/water wasused as a solvent system. The mixed solution of 10% (v/v) methanolsolution in water was applied first to elute, then A mixed solution of20% was applied, followed by a stepwise increase in increments of 10%(v/v) methanol concentration. A mixed solution of 10 ml was applied eachtime. The resultant eluted fractions were concentrated under reducedpressure at 35° C. to dryness, the dried fractions were dissolved in 10ml methanol, some of which (3, 30, 100 and 300 μl) was subjected to aWIPK induced activity assay. The assay procedure was as above.

Activity was detected in a fraction at 80% (v/v) methanol concentrationin a mixed solution of water/methanol, so this fraction was concentratedunder reduced pressure at 35° C. to dryness. The resultant dried residuewas dissolved in a small amount of mobile phase (methanol:water=4:1,v/v) and the dissolved substance was infused to a HPLC system fittedwith reverse phase high performance liquid chromatography (HPLC) column(LiChrospher 100RP-18, 5 μm particule size, 4 mm ID by 25-cm long,Hewlett Pachard). The above mobile phase was used as a solvent system,flow was at 1 ml/min flow rate, and the ultraviolet absorption wasmonitored and measured at the constant-wavelength of 254 nm. From 0.1minute after infusion, each of 3 ml aliquots of eluate were collected in90 fractions in total. Each fraction was concentrated under reducedpressure at 35° C. to dryness and each dried fractions were dissolved in10 ml methanol, some of which (3, 30, 100 and 300 μl) was subjected to aWIPK induced activity assay. The assay procedure was as above.

Activity was detected in the fractions eluted from 12.1 to 15.1 minutes,so this faction was further fractionated by the above HPLC column. Asolvent (Acetonitrile:water=3:2 (v/v)) was used as the solvent systemand flow was at 1 ml/min, and the ultraviolet absorption was monitoredand measured at the constant-wavelength of 254 nm. Activity was found inthe peak at 14.6 minutes retention time, so this peak was collected(FIG. 1).

This peak was used as an isolated WIPK activator for assay thereafter.

Example 2 Biological Activity Assay of Isolated Material

Isolated material was dissolved in dimethyl sulfoxide (DMSO) and dilutedwith 10 mM Mes-NaOH (pH 5.6) to an adequate concentration. If it wasused in an assay, the DMSO concentration was kept below 0.1%. Superiorhealthy leaves of 50-day-old tobacco (Nicotiana tabacum cv. Samsun NN)plant bodies were cut from the petiole, immediately put into a testtube, 10 mM Mes-NaOH (pH 5.6) solution containing isolated activesubstance of the invention at each concentration was added, and culturewas conducted at 24° C. 10 mM Mes-NaOH (pH 5.6) without active substanceof the invention was used as a control group. In a certain amount oftime, the leaves were collected and immediately frozen in liquidnitrogen to conduct WIPK activity measurement or RNA extraction forNorthern blot analysis. WIPK activity measurement, RNA extraction andNorthern blot analysis were performed following Seo et al. (1999) plantCell 11, 289-291. WIPK activity was measured 15 minutes after thesubject was allowed to absorb the isolated compound at the givenconcentration through the petiole. Only buffer was absorbed in a controlgroup. As another control group (healthy leaf), leaves were cut fromtobacco plant body to be immediately subjected to WIPK activitymeasurement. For proteinase inhibitor II (PI-II), basic PR-1, basic PR-2genes and ACO, transcription product amounts of PI-II, basic PR-1, basicPR-2 genes and ACO-coding gene were measured after allowing the subjectabsorb the isolated compound at the given concentration through thepetiole for a period of time. As a control group, only buffer wasabsorbed. The outline is as follows:

Nucleic acid hybridization reaction was performed on nylon membranecontaining 20 μg total RNA with ³²P-radiolabeled cDNA of tobacco PI-II,basic PR-1, basic PR-2 genes and ACO-coding gene. After the membrane waswashed it was used to expose to an X-ray film.

(Effects of Isolated Substance on JA and SA accumulation)

To evaluate whether the induction of basic pathogenesis-related proteingene (PR-1 and PR-2) and proteinase inhibitor II gene by syntheticsubstance occurred through JA or SA, 100 nM or 1 μM WAF-1 or water wasabsorbed into tobacco leaves through the petiole, followed by culture at24° C., to measure the intrinsic level of JA.

(Effects of Synthetic Substance on Ethylene Accumulation)

Ethylene acts as a signal in the induction of basic PR protein oftobacco plant, so the release of ethylene was measured after externallygiving WAF-1 to a tobacco leaf as above to evaluate the effects ofsynthetic substance on ethylene accumulation.

(Results)

(Effects of Isolated Substance on the Induction of WIPK Activity)

The isolated substance at each concentration shown in FIG. 2A was givento a leaf through the petiole to measure WIPK activity at 15 minutes,resulting in the induction of WIPK activity at any concentration. In thetreatment to a control group, little activity was observed.

Natural isolated substances at certain concentrations (1, 5 and 10 nM)or water as a control were given to leaves through the petiole as above,to measure WIPK activity (MBP phosphorylation activity) at 5, 15 and 30minutes. As a result, as shown in FIG. 2B, the induction of WIPKactivity was observed at 5 minutes, maximized at 15 minutes, andretained maximum activity at 30 minutes.

(Effects of Isolated Substance on Expression Induction of ProteinaseInhibitor II, Basic PR-1, Basic PR-2 Genes and ACO-Coding Gene)

The isolated substance at the concentration shown in FIG. 3 was given toleaves through the petiole to examine the accumulation of transcriptionproducts of proteinase inhibitor II gene in 2, 6 and 16 hours, resultingin the detection of accumulation at 16 hours. This amount wassignificantly more than that of accumulation in the control group. Theaccumulation of proteinase inhibitor II, basic PR-1, basic PR-2 and ACOsuggests that this active substance of the invention acts as a signaltransmitter of wound induction.

(Effects of isolated substance on the accumulation of JA and SA) Theamounts of JA and SA in leaves treated with 100 nM WAF-1 or 1 μM WAF-1were at the same level as those treated with water even after 3-, 6-,12- or 24-hours culture (data not shown), suggesting that external WAF-1does not induce any internal increase in JA or SA.

(Effects of Isolated Substance on Ethylene Accumulation)

As shown in the results, the levels of ethylene released from the leavestreated with 100 nM WAF-1 and 1 μM WAF-1 was 1.2 and 1.4 times higherthan the leaf treated with water, respectively. The results are shown inFIG. 11.

Example 3 Identification of WIPK Activator

The above WIPK activator was analyzed using NMR and MS. For NMRanalysis, JEOL JNM-A600 (JEOL. Ltd., Tokyo, Japan) was used. For MSanalysis, an Automass JMS-AM SUN (JEOL. Ltd., Tokyo, Japan) was used inEI mode (70 eV)

¹H and ¹³C NMRs were assigned by the spectral data analysis onPFG-DQFCOSY, PFG-HMQC and PFG-HMB. The amount of sample was small andthus ¹³C NMR spectrum could not be measured. Therefore, the chemicalshift level of ¹³C NMR was determined with 2D spectral data and assignedby comparison to the data reported on PFG-HMBC spectral data and therelated Labdan diterpenoid (Jikken-Kagaku-Koza 6, NMR (ExperimentalChemistry Course 6, NMR), 4^(th) edition, ed. The Chemical Society ofJapan, Maruzen, pp 99-176; Phytochemistry 40, 1213, 1995; Phytochemistry27, 624, 1988).

The results are shown in the following Table.

TABLE 1 WAF-1 (11E,13E)-labda-11,13-diene-8α,15-diol Proton δ ppm Carbonδ ppm

1α-H1β-H2-H₂3-H₂5-H6α-H6β-H7α-H7β-H9-H10-H11-H12-H15-H₂16-H₃0.86(m)1.38(m)**0.92(m)1.69(m)1.32(m)1.48(m)1.92(ddd, 12.7, 3.2,3.2)1.83(d, 10.7)5.69(dd, 15.6, 10.7)6.19(d, 15.6)5.64(t, 6.8)4.29(dd,6.8, 5.9)1.83(s) C-1C-2C-3C-4C-5C-6C-7C-8C-9C-10C-11C-12C-13C-14C-15C-1640.9*41.933.255.7*42.072.066.337.6125.7139.2135.8129.359.412.8 EI-MS m/z288 (M⁺—H₂O, 68%), 177(100), 133(60), 10 17-H₃ 1.20(s) C-17 25.2 68),95(66), 81(73), 69 (97), 43(0 18-H₃ 0.89(s) C-18 33.3 HR-EI-MS m/z[MH₂O]⁺: Found, 288.2453 19-H₃ 0.82(s) C-19 21.6 Calcd.forC20H32O288.2455 20-H₃ 0.94(s) C-20 15.9 15-OH 1.22(t, 5.9) *Unassigned Coupling constants (J in Hz) are given in parentheses.

As a result of the above, the structure of active substance of theinvention isolated in this invention was estimated as follows:

Example 4 Ascertainment of SIPK Activation Activity

Using the above WIPK activator, whether salicylate-induced proteinkinase (SIPK) was activated was examined.

As an active substance, the substance isolated in Example 1 was used asa standard. The isolated substance was dissolved in dimethyl sulfoxide(DMSO) and diluted to adequate concentration (0.3, 1.5 and 3 ng/ml) with10 mM Mes-NaOH (pH 5.6). If it was used in an assay, the DMSOconcentration was kept below 0.1%. Superior healthy leaves of 50-day-oldtobacco (Nicotiana tabacum cv. Samsun—NN) plant body was cut from thepetiole, immediately put into a test tube, 10 mM Mes-NaOH (pH 5.6)solution containing isolated active substance of the invention at eachconcentration was added, and cultured at 24° C. 10 mM Mes-NaOH (pH 5.6)without active substance of the invention was used as a control group.In a certain amount of time, the leaves were collected and immediatelyfrozen in liquid nitrogen to measure the SIPK activity. The measurementwas conducted as follows:

Crude protein of 50 μg and anti-SIPK antibody were reacted and themyelin basic protein (MBP) phosphorylation activity of SIPK in theresultant immune complex was measured.

(Results)

(Effects of Isolated Substance on the Induction of SIPK Activity)

The isolated substance at each concentration shown in FIG. 4 was givento a leaf through the petiole to measure the SIPK activity in 15minutes, resulting in the induction of SIPK activity at anyconcentration. In the control group, little activity was observed.

Therefore, it became clear that the isolated substance that induces WIPKactivity had the activity to induce SIPK activity also.

Natural isolated substances at certain concentrations (1, 5 and 10 nM)or water as a control were then given to leaves through the petiole tomeasure SIPK activity (MBP phosphorylation activity) in 5, 15 and 30minutes. As a result, as shown in FIG. 2B, it was found that theinduction of SIPK activity was already maximized at 15 minutes as shownfor WIPK activity.

Therefore, it was indicated that the isolated substance that inducesWIPK activity also had the activity to induce WIPK activity and toinduce SIPK activity in an extremely rapid, probably commom, mechanism.

Example 5 Synthesis of WIPK Activator and Ascertainment of the Effectsof the Synthetic Substance

To ascertain whether the substance having the structural formula asshown above has WIPK activation activity, a compound having the samestructure was synthesized. The synthetic scheme is shown below:

(Intermediate 2A)

Commercial sclareolide having the following structural formula:

(5.0 g, 20.0 mmol) was dissolved in anhydrous Et₂O (150 mL), followed bydropwise addition of 1.14 M Et₂O (19.3 ml, 22.0 mmol) of MeLi for 10-15minutes under nitrogen airflow with ice-cold stirring. After stirring atthe same temperature for 30 minutes, 10% H₂SO₄ (25 ml) was added to themixture solution, and stirred for 5 minutes to extract the reactionmixture with Et₂O. The organic layer was washed with 1% aqueous NaOHsolution, water and saturated brine, progressively, and dried overNa₂SO₄. The solvent was removed under reduced pressure and the resultingresidue was chromatographed over silica gel to abtain an intermediate 2A(4.8 g, 90%) from the elution with hexane-Et₂O (3:2) (colourlessacicular, melting point {mp} 64-65° C. {hexane}).

¹H-NMR δH (300 MHz) 0.79 (2×3H, s), 0.88 (3H, s), 1.11 (3H, S,>C(OH)CH₃), 2.20 (3H, S, —C(O)CH3), 2.44 (1H, dd, J=17.5 and 4.4 Hz,11-H), 2.54 (1H, dd, J=17.5 and 5.6 Hz, 11-H).

¹³C-NMR δC (75 MHz) 15.62, 18.33, 20.55, 21.35, 23.06, 30.23, 33.18,33.28, 38.26, 39.53, 41.69, 44.56, 55.80, 55.89, 73.08, 210.20.

(Intermediate 4A)

Intermediate 2A (1.00 g, 3.75 mmol) was dissolved in anhydrous CH₂Cl₂(20 ml) and-added with m-chloroperbenzoic acid (1.43 g, 8.27 mmol) undernitrogen airflow with ice-cold stirring, followed by stirring at thesame temperature for 1 hour, and additionally incubated at roomtemperature for 5 days. After the solvent was removed, the residue wasdissolved in MeOH solution (12 mL) containing 10% KOH and stirred atroom temperature for 24 hours. The reaction mixture was extracted byEt₂O and the Et₂O layer was washed with saturated NaHCO₃ solution, waterand saturated brine, progressively, and dried over Na₂SO₄. The solventwas removed under reduced pressure and the resulting residue wassubjected to flash chromatography to obtain an intermediate 4A (493 mg,55%) from the elution with hexane-AcOEt (3:1) (colourless acicular, mp11.8-119° C. {Et₂O-hexane}).

¹H-NMR δH (300 MHz) 0.79 (2×3H, s), 0.88 (3H, s), 1.35 (3H,s, >C(OH)CH3), 3.91 (2H, —CH2-).

¹³C-NMR ″C (75 MHz) 15.03, 18.60, 20.18, 21.62, 24.29, 33.27, 33.55,37.52, 40.00, 41.69, 44.45, 55.92, 60.50, 61.10, 75.07.

(Intermediate 5A)

Intermediate 4A (199 mg, 0.829 mmol), N-methylmorpholine N-oxide (292mg, 2.50 mmol), and 4A molecular sieve (524 mg) were suspended inanhydrous CH₂Cl₂ (5 ml) and added with perruthenate tetrapropylammonium(29 mg, 0.083 mmol) under nitrogen airflow with ice-cold stirring,followed by stirring at the same temperature for 10 minutes,additionally stirred at room temperature for 25 minutes. To the reactionmixture, Et₂O (20 ml) was added and stirred, followed by filtration withsilica gel. After the filtrate was concentrated, it was subjected toflash chromatography to obtain an intermediate 5A (126 mg, 64%) from theelution with hexane-Et₂O (10:1) (colourless wax solid).

¹H-NMR δH (300 MHz) 0.84 (3H, s), 0.90 (3H, s), 1.12 (3H, s), 1.39 (3H,s, >C(OH)CH3), 2.08 (1H, d, J1.4 Hz, 9-H), 10.03 (1H, d, J1.4 Hz, —CHO).

¹³C-NMR δC (75 MHz) 17.53, 18.14, 19.85, 21.33, 25.28, 33.22, 33.30,37.34, 39.77, 41.59, 42.67, 55.14, 71.26, 72.77, 208.16.

(Intermediate 6A)

NaNH₂ (30.5 mg, 0.781 mmol) was suspended in anhydrous THF (4.1 ml),followed by dropwise addition of3-ethoxycarbonyl-2-methyl-prop-2-enylphosphonate (0.20 ml, 0.824 mmol)

under nitrogen airflow with ice-cold stirring, and stirred at the sametemperature for 20 minutes. The reaction mixture was cooled to −78° C.,followed by the dropwise addition of a solution of intermediate 5A (61.1mg, 0.256 mmol) in THF (4.1 ml), stirred at −50° C. for 41 hours, thenback to room temperature, water added, and extraction with Et₂O. TheEt₂O layer was washed with water and saturated brine, progressively, anddried over Na₂SO₄. The solvent was removed under reduced pressure and aresulting residue was subjected to flash chromatography to abtain themixture of intermediates 6Ac and 6Ad (24 mg) and that of 6Aa and 6Ab (64mg) from the elution of hexane-AcOEt (10:1) solution. For the mixture ofintermediates 6Aa and 6Ab, intermediates 6Aa (8.9 mg, 10%) and 6Ab (52mg, 59%) were abtained by high performance liquid chromatography[elution solvent: hexane-AcOEt (5:1), elution rate: 10 ml/min], while,for the mixture of intermediates 6Ac and 6Ad, 6Ad (4.7 mg, 5.3%) and 6Ac(17 mg, 19%) were obtained from the elution site of exane-AcOEt (10:1)by additional flash chromatography.

The measured data on each intermediate of 6Aa-6Ad is shown below:

(Intermediate 6Aa)

Colourless Wax Solid

¹H-NMR δH (300 MHz) 0.84 (3H, s), 0.89 (3H, s), 0.96 (3H, s) 1.23 (3H,s, 17-H3), 1.28 (3H, t, J7.1 Hz, —OCH2-CH3), 2.30 (3H, d, J1.0 Hz,16-H3), 4.17 (2H, q, J7.1 Hz, —OCH2-CH3), 5.74 (1H, s, 14-H), 6.12 (1H,dd, J15.4 and 9.5 Hz, 11-H), 6.22 (1H, d, J15.4 Hz, 12-H).

¹³C-NMR δC (75 MHz) 14.07, 14.33, 15.98, 21.59, 25.23 and 33.38 (C-16,-17, -18, -19 and -20, and —OCH2-CH3) 18.40, 20.09, 40.96, 41.88 and42.33 (C-1, -2, -3, -6 and -7), 33.32 and 37.84 (C-4 and -10), 55.74 and66.48 (C-5 and -9), 59.72 (—OCH2-CH3), 72.21 (C-8), 119.07, 133.02 and138.77 (C-11, -12 and -14), 151.35 (C-13), 167.11 (C-15).

(Intermediate 6Ab)

Colourless Viscous-oily Substance

¹H-NMR δH (300 MHz) 0.82 (3H, s), 0.89 (3H, s), 0.95 (3H, s), 1.22 (3H,s, 17-H3), 1.27 (3H, t, J7.1 Hz, —OCH2-CH3), 2.02 (3H, d, J1.0 Hz,16-H3), 4.16 (2H, q, J7.1 Hz, —OCH2-CH3), 5.66 (1H, s, 14-H), 6.10 (1H,dd, J15.7 and 10.3 Hz, 11-H), 7.58 (1H, d, J15.7 Hz, 12-H).

¹³C-NMR δC (75 MHz) 14.32, 16.02, 21.33, 21.59, 25.01 and 33.39 (C-16,-17, -18, -19 and -20, and —OCH2-CH3) 18.40, 20.14, 40.91, 41.89 and42.64 (C-1, -2, -3, -6 and -7), 33.31 and 37.82 (C-4 and -10), 55.69 and66.62 (C-5 and -9), 59.73 (—OCH2-CH3), 72.21 (C-8), 117.01, 132.93 and134.29 (C-11, -12 and -14), 150.07 (C-13), 166.15 (C-15).

(Intermediate 6Ac)

Colourless acicular, mp 123-126° C. (hexane)

¹H-NMR δH (300 MHz) 0.86 (3H, s), 0.89 (3H, s), 1.04 (3H, s), 1.07 (3H,s), 1.28 (3H, t, J7.1 Hz, —OCH2-CH3), 2.31 (3H, d, J1.0 Hz, 16-H3), 4.17(2H, q, J7.1 Hz, —OCH2-CH3), 5.71 (1H, s, 14-H), 6.05 (1H, d, J15.6 Hz,12-H), 6.28 (1H, dd, J15.6 and 10.0 Hz, 11-H).

¹³C-NMR δC (75 MHz) 14.11, 14.35, 16.01, 21.80, 31.62 and 33.53 (C-16,-17, -18, -19 and -20, and —OCH2-CH3), 18.20, 18.31, 40.75, 42.01 and42.39 (C-1, -2, -3, -6 and -7), 33.43 and 38.25 (C-4 and -10), 55.57 and63.65 (C-5 and -9), 59.61 (—OCH2-CH3), 72.27 (C-8), 118.08, 134.66 and137.38 (C-11, -12 and -14), 152.26 (C-13), 167.27 (C-15).

(Intermediate 6Ad)

Colourless acicular, mp 115-117° C. (hexane)

¹H-NMR δH (300 MHz) 0.86 (3H, s), 0.88 (3H, s), 1.06 (3H, s), 1.07 (3H,s), 1.28 (3H, t, J7.1 Hz, —OCH2-CH3), 2.03 (3H, d, J1.1 Hz, 16-H3), 4.16(2H, q, J7.1 Hz, —OCH2-CH3), 5.63 (1H, s, 14-H), 6.28 (1H, dd, J15.9 and10.1 Hz, 11-H), 7.52 (1H, d, J15.9 Hz, 12-H).

¹³C-NMR δC (75 MHz) 14.33, 16.07, 21.30, 21.79, 31.69 and 33.52 (C-16,-17, -18, -19 and -20, and —OCH2-CH3) 18.20, 18.32, 40.63, 41.98 and42.41 (C-1, -2, -3, -6 and -7), 33.41 and 38.14 (C-4 and -10), 55.50 and63.67 (C-5 and -9), 59.56 (—OCH2-CH3), 72.38 (C-8), 115.97, 131.57 and136.17 (C-11, -12 and -14), 151.03 (C-13), 166.40 (C-15).

(Synthesis of WAF-1)

Then, of the above compounds, 6Aa-6Ad,

(10 mg, 0.029 mmol) was dissolved in anhydrous CH₂Cl₂ (0.5 ml), followedby dropwise addition of 1.0 M CH₂Cl₂ solution of diisobutylaluminumhydride (0.20 ml, 0.20 mmol) in nitrogen airflow at −78° C. understirring. After stirring at the same temperature for 30 minutes, andremoval of the refrigerant, the mixed solution was stirred for 30minutes with a gradual increase to room temperature. While on ice, AcOEt(0.5 ml), CH₂Cl₂ (5 ml), 0.5 M aqueous solution of sodium potassiumtartrate (1.1 ml) was added tp the reaction mixture, stirred at roomtemperature for 15 minutes, and extracted with Et. The organic layer waswashed with water and saturated brine, progressively, and dried overNa₂SO₄. The solvent was removed under reduced pressure and the resultingresidue was subjected to flash chromatography to abtain WAF-1 (9.0 mg,100%) from the elution site of hexane-AcOEt (1:1) (colourlessviscous-oily substance).

¹H-NMR δH (600 MHz) 0.82 (3H, s, 19-H3), 0.85 (1H, m, 1a-H), 0.88 (3H,s, 18-H3), 0.93 (1H, m, 5-H), 0.94 (3H, s, 20-H3), 1.13 (1H, m, 3a-H),1.20 (3H, s, 17-H3), 1.32 (1H, m, 6b-H), 1.38 (1H, m, 1b-H), 1.38 (1H,m, 2a-H), 1.38 (1H, m, 3b-H), 1.48 (1H, ddd, J12.7, 12.7 and 3.3 Hz,7a-H), 1.56 (1H, m, 2b-H), 1.69 (1H, m, 6a-H), 1.83 (1H, d, J10.3 Hz,9-H), 1.83 (3H, s, 16-H3), 1.92 (1H, ddd, J12.7, 3.1 and 3.1 Hz, 7a-H),4.29 (2H, d, J6.8 Hz, 15-H2), 5.64 (1H, t, J6.8 Hz, 14-H), 5.69 (1H, dd,J15.6 and 10.3 Hz, 11-H), 6.19 (1H, d, J15.6 Hz, 12-H).

¹³C-NMR δC (150 MHz) 12.85 (C-16), 15.92 (C-20), 18.44 (C-2), 20.07(C-6), 21.60 (C-19), 25.23 (C-17), 33.32 (C-4) 33.40 (C-18), 37.66(C-10), 40.91 (C-1), 41.95 (C-3), 42.03 (C-7), 55.84 (C-5), 59.31(C-15), 66.38 (C-9), 72.00 (C-8), 125.71 (C-11), 129.46 (C-14), 135.94(C-13), 139.44 (C-12).

EI-MS m/z288 (M⁺-H2O, 68%), 177 (100), 133 (60), 109 (68), 95 (66), 81(73), 69 (97), 43 (63). HR-EI-MSm/z [M-H2O]⁺: Measured value, 288.2453.Calculated value about C₂₀H₃₂O, 288.2455.

It was confirmed that this compound had the same structure as naturalsubstances of the invention.

(Ascertainment of WIPK Activation Activity of the Synthetic Substance)

Using the synthetic substance thus synthesized, the WIPK activitymeasurement test was conducted as stated in Example 1. The results areshown in FIG. 5.

(Ascertainment of SIPK Activation Activity of the Synthetic Substance)

Using the synthetic substance thus synthesized, the SIPK activitymeasurement test was conducted as stated in Example 4. The results areshown in FIG. 6.

(Ascertainment of WIPK and SIPK Activities of Natural Substance of theInvention)

Using natural isolated substances, preparations at concentrations of 10and 100 pM, and 1 and 100 nM were prepared and these naturalpreparations or water were penetrated into tobacco leaf disks, followedby sampling at 30 minutes after treatment, to measure the MBPphosphorylation activity of WIPK and SIPK as described above. Theresults are shown in FIG. 7.

(Results)

As these results show, in natural isolated substances, it seems to bethat the activation of WIPK and SIPK is significant at higherconcentrations than 10 pM and reaches a maximum at a concentrationbetween 100 pM and 1 nM. Synthetic WAF-1 also showed a similar aspect.

Therefore, it was ascertained that the synthetic preparations and thenatural isolated substance of the present invention produced effects atthe same concentrations.

Example 6 Accumulation of an Endogenous Amount of Active Substance ofthe Invention After TMV Infection and Wounding

The accumulation of an endogenous amount of active substance of theinvention according to this invention after TMV infection and wound wasascertained.

(Quantification Method for Active Substance of the Invention)

2 g of a leaf tissue was ground with 20 ml cold 80% (v/v) methanol in aPolytron and allowed to stand at 4° C. for 1 hour for extraction. Theextract was centrifuged at 10,000×g for 10 minutes. The precipitate wassuspended in 10 ml cold 80% (v/v) methanol and centrifuged at 10,000×gfor 10 minutes to collect the supernatant. The resultant twosupernatants were collected and the water phase concentrated to at 35°C. under reduced pressure with a rotary evaporator. The resultant waterphase was adjusted to pH 7.5 with 1M phosphate buffer and fractionatedwith the same amount of ethyl acetate three times. The supernatant ethylacetate layer was dehydrated with anhydrous sodium sulfate andconcentrated to dryness at 35° C. under reduced pressure with a rotaryevaporator. The dried substance was dissolved in 3 ml 80% (v/v) methanoland passed through a C18 Sep-Pak cartridge column (Waters, the U.S.A.)equilibrated with 80% (v/v) methanol inadvance. The flow-throughfractions were collected and concentrated to dryness at 35° C. underreduced pressure with a rotary evaporator. The concentrated residue wasdissolved in 80% (v/v) methanol and the solution was subjected to highperformance liquid chromatography (LiChrospher 100RP-18.5-μm particlediameter, 4 mm internal diameter, 25 cm length, Hewlett Packard).Elution was conducted with methanol solution (methanol:water=4:1) (v/v)at 1 ml/min of flow rate. The solution eluted from 11 to 16.5 minutesretention time was collected. After this the solution collected wasconcentrated to dryness, the resultant residue was dissolved in 200 μlacetonitrile solution (acetonitrile:water=9:8 (v/v)), one-tenth amountof which was subjected to the above described high performance liquidchromatography. The elution was conducted-with acetonitrile solution(acetonyl:water=9:8 (v/v)) at 1 ml/min of flow rate. Ultravioletabsorption was monitored at the constant-wavelength of 238 nm to detectWAF-1. To obtain the recovery rate of active substance of the inventionin extraction and purification procedures, 300 ng Labdan a was added asan internal standard in the initial process of the extraction procedure.

The retention times of the active substance of the invention and Labdana were 24.5 and 29.5 minutes, respectively. The endogenous amount ofactive substance of the invention was calculated with the calibrationcurve produced by a known amount of active substance of the invention.All data was obtained by correction to the loss based on the recoveryrate of active substance of the invention in extraction and purificationprocedures (65-75%).

Labdan a was produced as follows: Specifically, it was abtained byreduction of intermediate 6Ac with diisobutylaluminum hydride.

In FIG. 7, the data was shown to produce a calibration curve of thecompound of this invention, which was isolated from tobacco. As statedabove, in the experiment to produce a calibration curve, tobacco leafdisks were penetrated with water and natural substance of the inventionat each concentration, followed by sampling at 30 minutes aftertreatment, to measure the MBP phosphorylation activity of WIPK and SIPKas stated above.

Labdan a is a product obtained in a synthetic process of activesubstance of the invention, showing the chromatography behavior similarto that of the active substance of the invention, but not present in thetobacco plant body. Based on these properties, Labdan a was used as aninternal standard.

(Accumulation of the Endogenous Amount of Active Substance of thisInvention After the Change from 20 to 30° C.)

Tobacco leaves were inoculated with TMV (10 μg/ml) or buffer (mockinoculation) was inoculated tobacco leaves. After inoculation, leafdisks of 3 cm in diameter were stamped out of the inoculated leaves andput into a transparent plastic box spread with filter papers moistenwith distilled water, which was cultured in an incubator at 30° C. for48 hours, then transferred to another incubator at 20° C. to induce ahypersensitive reaction, followed by sampling of the disks over time tobe used in the quantification for active substance of the invention.

The results are shown in FIG. 8A. Units are nano grams (ng) activesubstance of the invention per 1 g fresh weight of leaf. Each data pointindicates mean ± standard deviation of measurements in triplicate.

(Accumulation of the Endogenous Amount of Active Substance of theInvention After the Start of Culture at 20° C.)

Tobacco leaves were inoculated with TMV (2 μg/ml) or buffer (mockinoculation) was inoculated. After inoculation, leaf disks of 3 cm indiameter were stamped out of the inoculated leaves and were put into atransparent plastic box spread with filter papers moisten with distilledwater, which was cultured in an incubator at 20° C., followed bysampling of the disks over time to be used in the quantification foractive substance of the invention. The results are shown in FIG. 8B. Theunits are nano grams (ng) active substance of the invention per 1 gfresh weight of leaf. Each data point indicates mean± standard deviationof measurements in triplicate.

(Accumulation of the Endogenous Amount of Active Substance of theInvention After Wounding)

Tobacco leaves were cut to the size of about 5-mm-square with a razor.The leaf segments sampled over time were used for the quantification ofactive substance of the invention. The results are shown in FIG. 9. Theunits are nano grams (ng) active substance of the invention per 1 gfresh weight of leaf. Each data point indicates mean ± standarddeviation of measurements in triplicate.

(Results and Discussion)

WIPK activating substance was isolated from the tobacco leaves in whicha hypersensitive reaction was induced. Hypersensitive reaction is atypical example of the pathogenesis resistance reaction of plants, inwhich the plant sites infected with the pathogen positively die, andconfines pathogens, resulting in necrosis lesion, followed by inhibitionof the infection spreading to non-infected sites. It is thoght thatresistant genes responding to pathogens in plants are involved in thehypersensitive reaction, for example, in the case of tobacco resistanceto TMV, such an example includes TMV-resistant gene N.

It is known that WIPK is activated at the initial stage of thehypersensitive reaction to TMV infection. Therefore, it was investigatedwhether the content of WIPK activating substance of this inventionchanged in the hypersensitive reaction. The hypersensitive reaction toTMV infection of tobacco with N gene is temperature sensitivity so thatit dose not occur at 28° C. or higher when N genes are not working,while it does occur at 24° C. or lower where N genes are working. Usingthis characteristic, in this Example, TMV-infected leaves of Samsun NNtobacco (which has N gene) were cultured at 30° C., then at 20° C., toinduce the hypersensitive reaction.

When TMV-inoculated leaves cultured at 30° C. for 48 hours were shiftedto 20° C., the content of WIPK activating substance began to increase at3 hours as shown in FIG. 8A and reached a maximum at 24 hours. Thecontent of this substance was 43±9 and 189±45 ng/g in fresh weight (FW)in 0 and 24 hours, respectively. The first increase occurred before WIPKactivation (occurred 4 hours after the shift to 20° C.) and theappearance of necrotic lesions (occurred 8 hours after the shift to 20°C.). In mock-inoculated leaves, there was no correnponding increase(FIG. 8A).

Similarly, when TMV-inoculated leaves were cultured at 20° C. shortlyafter inoculation, an increase in content of WIPK active substance ofthe invention was observed (FIG. 8B; 26±6 ng/g FW shortly afterinoculation and 126±22 ng/g FW at 48 hours). The increase in content wasseen 24 hours earlier than the 30 to 32 hours when necrotic lesionappeared. A transient increase in the content of WIPK activatingsubstance was also observed in mock-inoculated leaves continuouscultured at 20° C., suggesting that physical wounding induces anincrease in content of the substance in this invention in vivo

To ascertain whether wounding induces an increase in WIPK activatingsubstance, the leaves were injured physically, followed by monitoring ofthe quantitative variation of the substance. As a result, as shown inFIG. 9, the content of WIPK activating substance increased by 1.3-foldat 30 minutes after wounding (in healthy plant leaves, 24 ng/g FW and32±4 ng/g FW in 30 minutes), to almost about 3-fold in 180 minutes (60±7ng/g FW) suggesting that WIPK activating substance also immediatelyresponds to physical stress.

Example 7 Transcription Factor Regulation by WAF-1

To elucidate the role of WAF-1 in HR signaling pathway and the woundsignaling pathway, the effects of WAF-1 on gene expression induced by HRand wounding were investigated.

As a result, the investors found that WAF-1 activated tobacco WIZZ geneencoding WRKY type transcription factor. The results are shown below:

WIZZ is a factor whose expression was induced 10 minutes after wounding(Hara K, et al., MGG, 263, 30 (2000)) and it is known that it can bedetected before TMV necrotic lesion (Yoda H, et al. MGG 267, 154(2002)).

The transition in expression of the WIZZ transcript after stimulationwith WAF-1 in this invention was ascertained using the following method.Leaf disks stamped out of tobacco leaves were penetrated with 1 nM WAF-1solution or water and collected at 30 minutes after penetrance to beused for total RNA extraction. Total RNA extraction followed the methoddescribed in Seo et al. Plant Cell 1999, 11, 289-299. Isolated total RNAwas subjected to RNA gel blotting analysis following the aboveprocedure. A fragment, whose sequence corresponded to bases 877-1211 inthe WIZZ cDNA (335 bp) was amplified, and used as a probe. As aninternal standard probe, actin cDNA (described in Seo et al. 2000 12,917-932) was also used. An RNA gel blot membrane containing 20 μg totalRNA per lane was analized.

As a result, as shown in FIG. 10, the level of WIZZ transcript washigher in leaf disks impregnated with WAF-1 than in those inpregnatedwith water, suggesting that externally supplied WAF-1 enhanced WIZZexpression. Therefore, it was indicated that WAF-1 in this invention hasa role in regulatin of the immediate response shortly after stress.

The fact that the internal WAF-1 level increases before appearance ofnecrotic lesions indicates that WAF-1 accumulation is the initial eventof hypersensitivity cell death in TMV-infected tobacco. It seems thatthe induction of WIZZ gene expression by WAF-1 is mediated by WIPK andSIPK activation. In fact, AtMPK3 and AtMPK6 are orthologs of WIPK andSIPK of Arabidopsis, respectively, and it was reported that activationof wach of these was involved in the expression of a set of genesencoding WPKY transcription factor (Asai T, et al., Nature 415, 977(2002)). Therefore, WAF-1 appears to be a signal compound for activationin the signaling pathway of the HR signal and the wound signal.

Labdan-type diterpene seems widely present in the plant kingdom, but thebiological role is little understood. Sclareol has the followingstructure:

and is a main Labdan-type diterpene present in plant. Sclareol is anintimate analog of sclareolide, a starting substance of chemicalsynthesis of WAF-1, as stated above and is of interest in associationwith the invention stated in this specification.

Sclareol has various pharmacological activities. Such activities includeinduction of apoptosis (Dimas K, Leukemia Res. 25: 449 (2001)), fungalgrowth inhibition (Bailey, J. A. et al., Nature 255, 328 (1975)), andplant growth inhibition (Cutler H. G. et al., Plant Cell Physiol. 18,711 (1977)), etc. Sclareol also induces the expression of the tobaccogene encoding ABC transporter involved in the secretion and excretion oftoxic drugs (Jasinski et al. Plant Cell 13, 1095 (2001)).

It is interesting to note that sclareol constitutes 10% of the leafsurface exudates of Nicotiana glutinosa (Bailey, J. A. et al. , J. Gen.Microbiol. 85, 57 (1974)).

The inventors put tobacco leaves under reduced pressure, then returnedthe tobacco leaves back to atmospheric pressure, centrifuged them afterwater infiltration, followed by collection of the supernatant, toinvestigate the localization in tissue of WAF-1, resulting in collectionfrom the intercellular space of injured tobacco leaves, suggesting thatWAF-1 accumulates in the intercellular space after wounding. Therefore,sclareol and WAF-1, which are both low molecular weight compounds,appear to cooperate as natural defensive mechanisms in the surface andintercellular space of plant leaves.

Example 8 Corroboration of the Damage Resistance Enhancement in Plant byLabdan-typed Diterpenoid

It was indicated that the active substance of the invention obtained inthis invention affected the expression of the gene encoding proteinaseinhibitor II, an inhibitor of digestive enzymes in insect. Therefore, anexperiment was conducted by administering this substance to plants andpresenting vermin to the administered plant. In this example, rice wasused as a target. Growth was regulated in the rice administered withthis substance more than in the rice not administered.

Example 9 Defense from Stress

The active substance obtained in this invention was isolated fromTMV-infected tobacco in which hypersensitive reaction was induced. Thehypersensitive reaction confines pathogens because the infected cells inplant positively die, followed by an inhibition of pathogen growth,which is a typical example of the pathogenesis resistance reaction. Withthe above experiment, in the hypersensitive reaction induced with TMV,it was indicated that the endogenous amount of the substance at thisinvention began to increase at the initial stage of the reaction,suggesting that the substance in this invention plays an important rolein hypersensitive reaction.

In this Example, the substance in this invention was administered tonon-TMV-infected tobacco as a target. TMV stimulation was then given,under the same conditions as those in Example 1, to plants administeredwith the substance of this invention at the concentrations shown in FIG.12 and tp control plant not administered to measure the size of necroticlesions and the amount of TMV coat protein. The results are shown inFIG. 12.

(Results)

From these results, it was indicated that, in plants administered withthe substance of this invention, the size of necrotic lesions decreasedat concentrations higher than 100 pM and was dose-dependent. A decreasein TMV coat protein amount was also indicated, suggesting that, in thetobacco leaf treated with the substance in this invention, TMVreplication was inhibited. Therefore, it was indicated that theresistance to TMV increased in the plant administered with the substancein this invention more than in the plant not administered, whichcorroborated that the substance in this invention gave stress resistanceto plants.

Example 10 Promotion of Auxetic Growth in Plants by WAF-1

By administration of the compound of this invention, ACO wasaccumulated, so that the effects of the compound in this invention onauxetic growth was investigated.

(Plant Material)

In this Example, 15-day-old Arabidopsis (Arabidopsis thaliana),1-month-old tobacco (Nicotiana tabacum cv. Samsun—NN), or 20-day-oldrice (Oryza sativa) was used to provide plant bodies. The compound ofthis invention was dissolved in dimethyl sulfoxide (DMSO) and dilutedwith 10 mM Mes-NaOH (pH 5.6) to an adequate concentration (300, 100 and30 ng/ml). 10 mM of Mes-NaOH containing the compound of this inventionat the given concentration was sprayed to tobacco and rice plant bodies,followed by culture at 24° C. As a control group, 10 mM Mes-NaOH (pH5.6) free of the compound of this invention was used. In a certainamount of time, the periphery and the height of stem above ground levelwere measured in rice, Arabidopsis and tobacco plant bodies.

(Result)

From these results, in comparison with the control group, a delay ingrowth of the periphery and height of stem was observed in Arabidopsisand tobacco plant bodies and the growth of height above ground level wasobserved in rice plant body in those plants administered with thecompound of this invention. This indicates that the compound in thisinvention is involved in the growth of the periphery and the height inplant bodies and useful in regulating the growth of plant body.

Example 11 Effects of WAF-1 on Flowering

The administration of the compound in this invention induces theaccumulation of ethylene in plant tissue so the effects of the compoundof this invention on flowering was investigated.

(Plant Material)

In this example, 1-month-old petunia plant body was used. The compoundof this invention was dissolved in dimethyl sulfoxide (DMSO) and dilutedwith 10 mM Mes-NaOH (pH 5.6) to an adequate concentration (300, 100 and30 ng/ml). 10 mM Mes-NaOH containing the compound of this invention atthe given concentration was sprayed on the petunia plant bodies,followed by culture at 24° C. In a control group, 10 mM Mes-NaOH (pH5.6) without the compound of this invention was used. After a certainamount of time, observation of the gross flowering features of the plantbodies was performed. The administration of the compound of thisinvention to plants was then discontinued, only buffer was sprayed, andthe flowering was observed.

(Results)

From these results, compared with a control group, a delay in floweringwas observed in the petunia plant bodies administered with the compoundof this invention, and that the discontinuation of administration led toobservation of flowering, suggesting that the compound of this inventionwas useful at regulating flowering of plant bodies.

As described above, this invention is exemplified using the preferredexamples of this invention. It is understood that the range of thisinvention should be interpreted only by the claims. It is understoodthat the content of other patents, patent applications and literaturescited in this specification should be herein incorporated by referencein their entitity as if the content itself is specifically described inthis specification.

1. A compound having the following structure:

wherein, in the formula: X is selected from the group consisting ofhydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; oneof Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Z is asingle bond, or a divalent group having alkane or substituted alkanehaving two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and R¹-R²⁴ areindependently selected from the group of hydrogen, alkyl, andsubstituted alkyl.
 2. The compound of claim 1, wherein one of Y¹ and Y²is hydrogen, and the other is a methylol, substituted methylol,C1-aldehyde, C1-carboxyl, or substituted C1-carboxyl group.
 3. Thecompound of claim 1, wherein all of R¹-R²⁴ are hydrogen.
 4. The compoundof claim 1, wherein X is hydroxy.
 5. The compound of claim 1, having thefollowing structural formula:


6. A composition, comprising a compound having the following structure:

wherein, in the formula: X is selected from the group consisting ofhydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; oneof Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Z is asingle bond, or a divalent group having alkane or substituted alkanehaving two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and R¹-R²⁴ areindependently selected from the group of hydrogen, alkyl, andsubstituted alkyl.
 7. A composition for imparting stress resistance to aplant or augmenting said stress resistance, comprising a compound havingthe following structure:

wherein, in the formula: X is selected from the group consisting ofhydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; oneof Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Z is asingle bond, or a divalent group having alkane or substituted alkanehaving two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and R¹-R²⁴ areindependently selected from the group of hydrogen, alkyl, andsubstituted alkyl.
 8. The composition of claim 7, wherein said stressresistance is at least one resistance selected from the group consistingof wound resistance, insect resistance, disease resistance, andhypersensitivity cell death resistance.
 9. The composition of claim 7,wherein the imparting or augmenting of said stress resistance isaccomplished by controlling the activity of at least one proteinselected from the group consisting of wound-induced protein kinases,salicylic acid-induced protein kinases, pathogenesis-related proteins,and 1-amino-cyclopropane-t-carboxylic acid synthetases.
 10. Thecomposition of claim 7, wherein the imparting or augmenting of saidstress resistance is accomplished by controlling at least one signalingsystem selected from the group consisting of jasmonic acid signalingsystems and salicytic acid signaling systems.
 11. A method of impartingstress resistance to a plant or augmenting said stress resistance,wherein said method comprises the following steps: a) applying to saidplant a compound having the following structure:

wherein, in the formula: X is selected from the group consisting ofhydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; oneof Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Z is asingle bond, or a divalent group having alkane or substituted alkanehaving two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and R¹-R²⁴ areindependently selected from the group of hydrogen, alkyl, andsubstituted alkyl.
 12. The method of claim 11, wherein said stressresistance is at least one resistance selected from the group consistingof wound resistance, insect resistance, disease resistance, andhypersensitivity cell death resistance.
 13. The method of claim 11,wherein the imparting or augmenting of said stress resistance isaccomplished by controlling the activity of at least one proteinselected from the group consisting of wound-induced protein kinases,salicylic acid-induced protein kinases, pathogenesis-related proteins,and 1-amino-cyclopropane-t-carboxylic acid synthetases.
 14. The methodof claim 11, wherein the imparting or augmenting of said stressresistance is accomplished by controlling at least one signaling systemselected from the group consisting of jasmonic acid signaling systemsand salicylic acid signaling systems.
 15. A method of producing stressresistant plants, comprising: a) applying to said plant a compoundhaving the following structure:

wherein, in the formula: X is selected from the group consisting ofhydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; oneof Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Z is asingle bond, or a divalent group having alkane or substituted alkanehaving two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and R¹-R²⁴ areindependently selected from the group of hydrogen, alkyl, andsubstituted alkyl.
 16. A method of producing stress resistant planttissues, comprising: a) applying to said plant tissue a compound havingthe following structure:

wherein, in the formula: X is selected from the group consisting ofhydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; oneof Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Z is asingle bond, or a divalent group having alkane or substituted alkanehaving two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and R¹-R²⁴ areindependently selected from the group of hydrogen, alkyl, andsubstituted alkyl.
 17. A method of producing stress resistant plantcells, comprising: a) applying to said plant cell a compound having thefollowing structure:

wherein, in the formula: X is selected from the group consisting ofhydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; oneof Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Z is asingle bond, or a divalent group having alkane or substituted alkanehaving two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and R¹-R²⁴ areindependently selected from the group of hydrogen, alkyl, andsubstituted alkyl.
 18. A method of producing stress resistant plantseeds, comprising: a) applying to said plant a compound having thefollowing structure:

wherein, in the formula: X is selected from the group consisting ofhydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; oneof Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Z is asingle bond, or a divalent group having alkane or substituted alkanehaving two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and R¹-R²⁴ areindependently selected from the group of hydrogen, alkyl, andsubstituted alkyl; and b) obtaining said seed from said plant.
 19. Amethod of synthesizing a compound having the following structure:

wherein, in the formula: X is selected from the group consisting ofhydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; oneof Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Z is asingle bond, or a divalent group having alkane or substituted alkanehaving two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and R¹-R²⁴ areindependently selected from the group of hydrogen, alkyl, andsubstituted alkyl, said method comprises the following steps: a)reacting a compound (an intermediate 1) having

wherein, in the formula: R⁵-R²⁴ are independently selected from thegroup consisting of hydrogen, alkyl, and substituted alkyl, and the sameas R¹-R²⁴ for WAF, with an alkyl lithium to provide an intermediate 2;

b) mixing and reacting the product obtained in a) withm-chloroperbenzoic acid and then with a 10% potassium hydroxide inmethanol to provide an intermediate 4;

c) reacting the product obtained in b) with N-methylmorphorine N-oxidein the presence of tetrapropyl ammonium peruthenate to provide anintermediate 5;

d) adding a compound

wherein, one of V¹ and V² is hydrogen or alkyl, and the other is Z-V,and wherein Z is (CH2)n-C(═O)—O—, V is alkyl, n is an integer of 0 ormore, and R is alkyl, to said intermediate 5 obtained in the step c) inan organic solvent in the presence of NaNH₂ to provide an intermediate(6):

e) adding diisobutyl aluminum hydride in an organic solvent to saidintermediate (6) obtained in the step d) to provide

wherein, X is selected from the group consisting of hydroxy, substitutedhydroxy, halogen, thiol, and substituted thiol; one of U¹ and U² ishydrogen or alkyl, and the other is Z-U, wherein Z is a single bond, ora divalent group having alkane or substituted alkane having two hydrogenatoms removed, and U is hydroxy; and R¹-R²⁴ are independently selectedfrom the group of hydrogen, alkyl, and substituted alkyl; and optionallya further oxidation or substitution step where Y¹ is other than hydroxy.20. The method of claim 19, wherein said X is hydroxy; one of said Y¹and Y² is hydrogen, and the other is methylol; all of R¹-R²⁴ arehydrogen; said organic solvent is THF; the alkyl lithium in said step a)is methyl lithium; one of U¹ and U² is hydrogen, and the other is Z-U,wherein Z is —CH₂—, and U is hydroxy; and one of said V¹ and V² ishydrogen, and the other is —C(═O)—O—CH₂CH₃.
 21. A method of quantifyinga compound having the following structure:

wherein, in the formula: X is selected from the group consisting ofhydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; oneof Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Z is asingle band, or a divalent group having alkane or substituted alkanehaving two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and R¹-R²⁴ areindependently selected from the group of hydrogen, alkyl, andsubstituted alkyl, said method comprises the following steps: 1)providing a sample; 2) adding the predetermined amount of the stericisomer of said compound to said sample; 3) separating said sample by areverse phase liquid chromatography; and 4) calculating the amount ofsaid compound from said separated steric isomer.
 22. The method of claim21, wherein said compound has the following structural formula:

said steric isomer has the following structural formula:


23. The method of claim 21, wherein said sample is extracted withmethanol and subsequently with methyl acetate, prior to the separationwith said reverse phase liquid chromatography.
 24. The method of claim21, wherein the separation with said reverse phase liquid chromatographycomprises a separation with a C18 reverse phase liquid chromatography,and said separation comprises a first separation in 80%:20% (v/v)methanol:water, and a separation with 9:8 (v/v) acetonitrile:water. 25.The method of claim 21, wherein said calculation comprises thecorrection of the recovery loss.
 26. A composition for inducing a rapidaccumulation of a WRKY family gene in a plant under a conditionrequiring the accumulation of a WRKY family gene, said compositioncomprises a compound having the following structure:

wherein, in the formula: X is selected from the group consisting ofhydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; oneof Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Z is asingle bond, or a divalent group having alkane or substituted alkanehaving two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and R¹ R²⁴ areindependently selected from the group of hydrogen, alkyl, andsubstituted alkyl.
 27. The composition of claim 26, wherein saidcompound has the following structural formula:


28. The composition of claim 26, wherein the condition requiring theaccumulation of said WRKY family gene is a condition requiring a rapidresponse to stress.
 29. The composition of claim 26, wherein said plantis provided with a resistance to wound resistance, insect resistance,disease resistance, and hypersensitivity cell death resistance byinducing a rapid accumulation of said WRKY family gene.
 30. Thecomposition of claim 26, wherein said WRKY family gene is WIZZ or TIZZ.31. A composition for regulating the expression of a WRKY family gene,comprising a compound having the following structure:

wherein, in the formula: X is selected from the group consisting ofhydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; oneof Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Z is asingle bond, or a divalent group having alkane or substituted alkanehaving two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and R¹-R²⁴ areindependently selected from the group of hydrogen, alkyl, andsubstituted alkyl.
 32. A method of inducing a rapid accumulation of aWRKY family gene in a plant under a condition requiring the accumulationof a WRKY family gene, wherein said method comprises the followingsteps: a) applying to said plant a compound having the followingstructure:

wherein, in the formula: X is selected from the group consisting ofhydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; oneof Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Z is asingle bond, or a divalent group having alkane or substituted alkanehaving two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and R¹-R²⁴ areindependently selected from the group of hydrogen, alkyl, andsubstituted alkyl.
 33. The method of claim 32, wherein said compound hasthe following structural formula:


34. The method of claim 32, wherein the condition requiring theaccumulation of said WRKY family gene is a condition requiring a rapidresponse to stress.
 35. The method of claim 32, wherein said plant isprovided with a wound resistance, insect resistance, disease resistance,and hypersensitivity cell death resistance by inducing a rapidaccumulation of said WRKY family gene.
 36. The method of claim 32,wherein said WRKY family gene is WIZZ or TIZZ.
 37. The method of claim32, wherein said compound is applied immediately after the accumulationof said WRKY family gene is required.
 38. A composition for regulatingthe expression of a WRKY family gene, comprising the compound ofclaim
 1. 39. A composition for facilitating the elongating growth orauxetic growth of a plant, inhibiting the elongating growth of a plant,facilitating the maturation of a plant, or regulating the flowering of aplant, said composition comprises a compound having the followingstructure:

wherein, in the formula: X is selected from the group consisting ofhydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; oneof Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Z is asingle bond, or a divalent group having alkane or substituted alkanehaving two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and R¹-R²⁴ areindependently selected from the group of hydrogen, alkyl, andsubstituted alkyl.
 40. A method of facilitating the elongating growth orauxetic growth of a plant, inhibiting the elongating growth of a plant,facilitating the maturation of a plant, or regulating the flowering of aplant, said method comprises applying to a plant a compound having thefollowing structure:

wherein, in the formula: X is selected from the group consisting ofhydroxy, substituted hydroxy, halogen, thiol, and substituted thiol; oneof Y¹ and Y² is hydrogen or alkyl, and the other is Z-W, wherein Z is asingle bond, or a divalent group having alkane or substituted alkanehaving two hydrogen atoms removed, and W is hydroxy, substitutedhydroxy, aldehyde, carboxyl, or substituted carboxyl; and R¹-R²⁴ areindependently selected from the group of hydrogen, alkyl, andsubstituted alkyl.